AGRICULTUR 


BIOLOGY 
;W 

G 


LABORATORY  MANUAL 

IN 

GENERAL  MICROBIOLOGY 


PREPARED    BY    THE 

LABORATORY  OF  BACTERIOLOGY  AND   HYGIENE 

MICHIGAN  AGRICULTURAL  COLLEGE 


FIRST    EDI T ION 
SECOND    THOUSAND 


NEW  YORK 

JOHN  WILEY  &  SONS,  INC. 

LONDON:   CHAPMAN  &  HALL,  LIMITED 

1916 


BiOLOGY 
R 
G 


COPYRIGHT,  1916 

BY 
WARD    GILTNER 


LIBRAY 


PRESS  OF 

BRAUNWOHTH    &   CO. 

BOOK  MANUFACTURERS 

BROOKLYN,   N.  Y. 


PREFATORY  NOTE 


LABORATORY  instruction  in  bacteriology  at  the  Michigan 
Agricultural  College  developed  under  the  direction  of  Dr. 
C.  E.  Marshall.  This  laboratory  guide  represents  the  accu- 
mulated efforts  of  instructors  working  for  a  period  in  excess 
of  a  decade.  To  Assistant  Professor  L.  Zae  Northrup  is 
due  the  credit  for  collecting  and  arranging  the  material  pre- 
sented as  well  as  for  preparing  de  novo  many  of  the  experi- 
ments and  much  of  the  supplementary  matter.  She  has 
been  assisted  by  Mr.  W.  L.  Kulp.  Dr.  E.  T.  Hallman  and 
Dr.  L.  R.  Himmelberger  have  taken  the  responsibility  for 
arranging  the  exercises  relating  to  immunity,  serum  therapy 
and  pathologic  bacteriology.  Great  praise  is  due  Dr.  F.  H. 
H.  Van  Suchtelen  for  introducing  many  new  features  into 
the  laboratory  work  during  the  academic  year  1912-13, 
and  also  Dr.  Otto  Rahn  for  his  several  years  of  admirable 
effort  immediately  preceding.  Others  whose  influence  has 
been  felt  in  creating  this  guide  and  to  whom  credit  is  due 
are  Professors  W.  G.  Sackett,  S.  F.  Edwards,  L.  D.  Bush- 
nell,  C.  W.  Brown  and  W.  H.  Wright. 

While  some  claim  to  originality  may  be  made  for  this 
laboratory  guide,  it  is  to  be  expected  that  much  of  the 
material  herein  has  been  presented  in  various  other  manuals 
and  perhaps  in  better  form  in  many  instances.  The  greatest 
effort  has  been  made  to  make  this  a  laboratory  guide  to 
General  Microbiology,  leaving  the  particular  fields  of  dairy, 
soil,  water,  medical  and  other  phases  of  bacteriology  to 
special  guides  already  in  print  or  at  present  projected.  The 
presentation  of  this  manual  to  the  public  is  in  no  way  an 

iii 


IV  PREFATORY  NOTE 

intimation  that  the  special  iields  have  not  been  admirably 
dealt  with  by  others. 

The  subject  matter  given  under  Part  I  of  this  manual  is 
primarily  for  the  purpose  of  giving  a  working  knowledge  of 
laboratory  methods  used  in  the  study  of  microorganisms. 
Molds,  yeasts  and  bacteria  are  taken  up  in  the  order  of  their 
comparative  sizes  and  studied  as  to  their  identification  by 
morphological  and  cultural  methods.  It  is  presupposed  that 
the  student  has  a  knowledge  of  these  microorganisms 
acquired  from  preceding  lectures  in  microbiology. 

Part  II  consists  of  exercises  demonstrating  the  various 
physiological  activities  of  microorganisms. 

Part  III  deals  with  applied  microbiology.  After  the 
student  has  familiarized  himself  with  the  ordinary  tools  and 
technic,  etc.,  as  dealt  with  in  Parts  I  and  II,  it  is  not  neces- 
sary that  he  be  burdened  with  minute,  detailed  instructions. 
We  have  had  this  in  mind  in  preparing  Part  III. 

No  attempt  has  been  made  to  compile  an  exhaustive 
list  of  exercises;  the  aim  has  been  only  to  cover  a  wide 
range  of  activities  under  each  different  subject.  In  many 
cases,  exercises  have  been  taken  directly,  with  few  or  no 
modifications,  from  laboratory  manuals  already  in  print. 
Credit  has  not  been  given  directly;  the  list  of  references, 
however,  includes  all  books  from  which  material  has  been 
taken. 

The  purpose  of  this  laboratory  manual  is  to  make  the 
student  more  independent.  Practically  all  directions  for 
work  to  be  done  are  contained  in  it;  for  this  reason  the  work 
as  assigned  from  day  to  day  should  be  read -over  carefully 
before  beginning  an  exercise  and  then  followed  step  by  step. 
Any  desirable  changes  in  directions  may  be  indicated  by 
the  instructor. 

WARD  GILTNER, 

Head  of  Department, 
EAST  LANSING,  MICH. 
Sept.  1,  1915. 


CONTENTS 


PART  I 

GENERAL  MORPHOLOGICAL  AND   CULTURAL 
METHODS 

PAGE 

Exercise  1.     Cleaning  Glassware 1 

Sterilization 5 

Exercise  2.     Preparation  of  Glassware  for  Sterilization 15 

Nutrient  Media 18 

Exercise  3.     Titration  of  Media 20 

Milk 23 

Exercise  4.     Preparation  of  Litmus  Milk 25 

Exercise  5.     Preparation  of  Glycerin  Potato 26 

Exercise  6.     Preparation  of  Meat  Infusion 28 

Exercise  7.     Preparation  of  Nutrient  Broth 29 

Gelatin 31 

Exercise  8.     Preparation  of  Nutrient  Gelatin 36 

Agar : . . .  37 

Exercise  9.     Preparation  of  Nutrient  Agar 41 

Exercise  10.     Preparation  of  Dunham  s  Peptone  Solution 43 

Exercise  11.     Nitrate  Peptone  Solution 44 

Cultures 44 

Exercise  12.     Preparation  of  Plate    Cultures,    Loop    or    Straight 

Needle  Dilution  Method 49 

Exercise  13.     Preparation  of  Plate  Cultures,  Quantitative  Dilu- 
tion Method 52 

Exercise  14.     Methods  of  Counting  Colonies  in  Petri  Dish  Cul- 
tures    56 

Exercise  16.     Isolation  of  Microorganisms  from  Plate  Cultures 

and  Method  of  Making  Agar  Streak  Culture.  ...  58 
Exercise  16.     Method  of  Making  Transfers  of  Pure  Cultures  into 

a  Liquid  Medium 60 

Exercise  17.     Method  of  Making  Stab  Cultures 61 

Exercise  18.     Preparation  of  a  Giant  Colony 61 

The  Microscope , . .  .  63 


VI 


CONTENTS 


PAGE 

Exercise  19.     Method  of  Measuring  Microorganisms 70 

Exercise  20.     Determination   of  the   Rate   of   Movement   of   Motile 

Organisms 74 

Exercise  21.     Preparation  of  a  Hanging  Drop 74 

Exercise  22.     Preparation  of  the  Adhesion  Culture 78 

Exercise  23.     Preparation  of  the  Moist-chamber  Culture 80 

Exercise  24.     Preparation  of  the  Agar  Hanging-block  Culture ....     81 
Exercise  25.     Lindner's  Concave-slide  Method  of  Demonstrating 

Fermentation 83 

Exercise  26.     Lindner's  Droplet  Culture 84 

Exercise  27.     Chinese  Ink  Preparation. . 85 

Exercise  28.     The  Staining  of  Microorganisms 87 

Exercise  29.     Anjeszky's  Method  of  Staining  Spores 90 

Exercise  30.     Method  of  Staining  Tubercle  and  Other  Acid-fast 

Bacteria 92 

Exercise  31.     Method  for  Staining  Flagella 93 

Exercise  32.     Gram's  Method  of  Staining 95 

Exercise  33.     Method  for  Staining  Capsules 97 

Exercise  34.     Method  of  Making  Impression  Preparations 98 

Exercise  36.     Method  of  Staining  the  Nuclei  .of  Yeast  Cells 99 

General  Characteristics  of  Mold  Growth 100 

Exercise  36.     Microscopical  Examination  of  Molds 105 

Exercise  37.     The  Study  of  Molds 107 

Exercise  38.     To  Determine  the  Acidity  Changes  Produced  by 

,  Molds  in  Cider 112 

Exercise  39.     To  Demonstrate  the  Pathogenic  Nature  of  Molds. .   113 

Yeasts - 114 

Exercise  40.     To  Isolate  a  Pure  Culture  of  Saccharomyces  Cere- 
visiaB  and  to  Study  the  Flora  of  a  Compressed 

Yeast  Cake 114 

Exercise  41.     Apparatus  and  Methods  for  the  Study  of  Gaseous 

Fermentation 117 

Exercise  42.     The  Study  of  Yeasts 119 

Exercise  43.     The  Study  of  Bacteria 125 

Exercise  44.     Ehrlich's  Method  of  Testing  Indol  Production 132 

Exercise  45.     Tests  for  the  Reduction  of  Nitrates 133 

Descriptive  Chart — Society  of  American  Bacteriologists 134 

Exercise  46.     To  Demonstrate    the    Efficiency    of   Intermittent 

Heating  as  a  Method  of  Sterilizing  Media 139 

Exercise  47.     To  Compare  Morphologically  Protozoa  with  Bac- 
teria     140 

Exercise  48.     To  Study  the  Natural  Decomposition  of  Milk 141 


CONTENTS 


Vll 


Exercise  49.     To  Isolate  Spore-forming   Bacteria  and  to  Study 

Spore  Formation 144 

Exercise  50.  To  Demonstrate  the  Efficiency  of  Filtration  as  a 

Means  of  Removing  Microorganisms  from  Liquids  145 
Exercise  61.  To  Demonstrate  the  Presence  of  Microorganisms  in 

Air,  on  Desk,  Floor,  etc 146 

Exercise  62.  Qualitative  Study  of  the  Microorganisms  of  the  Skin 

and  Hair , 148 

Exercise  53.  Qualitative  Study  of  the  Microorganisms  of  the 

Mucous  Membrane  ...  ....    150 


PART  II 

PHYSIOLOGY  OF  MICROORGANISMS 

Exercise  1.     To  Demonstrate  the  Small  Amount  of  Food  Needed 

by  Bacteria 152 

Some  Physiological  Classifications  of  Bacteria 153 

Anaerobic  Culture  Methods 155 

Exercise  2.     The   Effect   of   Anaerobic   Conditions  upon   Micro- 
organisms from  Manure 165 

Exercise  3.     To  Demonstrate  that  Acids  Are  Formed  from  Car- 
bohydrates by  Bacteria 167 

Exercise  4.     To  Show  that  Organic  Acids  May  Serve  as  a  Food 

for  Some  Organisms 169 

Exercise  5.     To  Demonstrate  the  Variation  in  Food  Requirements 

of  Bacteria 170 

Exercise  6.     To  Demonstrate  the  Splitting  of  Carbohydrates  into 

Alcohol  and  CO2 171 

Exercise  7.     To  Demonstrate  the  Necessity  of  Nitrogen  in  Some 

Form  for  Microbial  Growth 172 

ExerciseS.     To  Demonstrate  the  Production  of  H2S  by  Bacteria.    174 
Exercise  9.     The  Effect  of  Physical  and  Chemical  Agencies  on 

Microbial  Pigment 175 

Exercise  10.     To    Illustrate   One    of   the   Physical    Products   of 

Metabolism 177 

Enzymes:     Classifications  and  Reactions 178 

Exercise  11.     A  Comparison  of  Acid  and  Rennet  Curds 185 

Exercise  12.     To  Show  the  Action  of  Proteolytic  Enzymes  upon 

Gelatin .  .  .187 


Vlll 


CONTENTS 


Exercise  13.   -  To  show  the  Action  of  Proteolytic    Enzymes   upon 

Casein. 188 

Exercise  14.     To  Show  the  Action  of  Enzymes  upon  Starch 189 

Exercise  16.     To  Show  the  Action  of  Reducing  Enzymes 190 

Exercise  16.     To  Show  the  Action  of  the  Enzyme  Catalase 191 

Exercise  17.     To  Demonstrate  the  Oxidizing  Enzyme  of  Vinegar 

Bacteria 192 

Exercise  18.     To  Demonstrate    the  Necessity  for  an  Activator 

for  the  Enzymic  Action  of  Rennet 193 

Exercise  19.  Effect  of  Concentrated  Solutions  upon  Microor- 
ganisms    195 

Exercise  20.     The  Effect  of  Desiccation  upon  Bacteria 197 

Exercise  21.  The  Determination  of  the  Optimum,  Maximum 
and  Minimum  Temperature  Requirements  for 
Certain  Organisms 198 

Exercise  22.     The   Effect   of   Freezing  upon  Spore-forming  and 

Non-spore-forming  Bacteria 199 

Exercise  23.  The  Determination  of. the  Thermal  Death  Point  of 
a  Spore-forming  and  a  Non-spore-forming  Or- 
ganism   200 

Exercise  24.     To  Determine  the  Relative  Effect  of  Moist  and 

Dry  Heat  on  Bacteria 202 

Exercise  25.     To  Determine  the  Effect  of  Pasteurization  upon  the 

Growth  of  Microorganisms 203 

Exercise  26.     To  Illustrate  the  Effect  of  the  Reaction  of  the 

Nutrient  Medium  upon  Microorganisms .  : 205 

Exercise  27.     To  Determine  the  Effect  of  Diffused  Light  upon 

Molds 206 

Exercise  28.     To  Show  the  Influence  of  Direct  Sunlight  upon  the 

Growth  of  Microorganisms 208 

Exercise  29.     Determination  of  the  Phenol  Coefficient  of  Some 

Common  Disinfectants 210 

Exercise  30.     To  Determine  the  Action  of  Formaldehyde  upon 

the  Microflora  of  Milk 213 

Exercise  31.     To  Illustrate  Symbiosis 214 

Exercise  32.  To  Illustrate  One  of  the  Phases  of  Mutual  Rela- 
tionship of  Microorganisms 215 

Exercise  33.  To  Demonstrate  the  Effect  of  the  Metabolic  Prod- 
ucts of  Bact.  Lactis  Acidi  on  its  Activities 217 


CONTENTS 


IX 


PART   III 
APPLIED   MICROBIOLOGY 

AIR  MICROBIOLOGY 
Exercise  1.     Quantitative  Bacterial  Analysis  of  Air .  .  . 


PAGE 

.  220 


WATER  AND  SEWAGE  MICROBIOLOGY 

Exercise  1.     Bacteriological   Analysis  of  Water   from   a   Source 

Suspected  of  Sewage  Contamination 223 

Exercise  2.  Bacteriological  Analysis  of  Water  Suspected  of 

Sewage  or  Other  Pollution 228 

Exercise  3.  To  Demonstrate  the  Efficiency  of  Chloride  of  Lime 

as  an  Agent  in  the  Purification  of  Drinking  Water .  236 
Exercise  4.  To  Demonstrate  the  Efficiency  of  the  Berkefeld 

Filter  Candle  as  a  Means  of  Water  Purification . . .  238 

SOIL  MICROBIOLOGY 

Exercise  1.     To  Test  the  Calalytic  Power  of  Soil 239 

Exercise  2.  A  Comparative  Study  of  the  Number  and  Types  of 

Microorganisms  in  Soil 241 

Exercise  3.  To  Illustrate  the  Effect  of  Aeration  of  Soils  on  the 

Activities  of  Microorganisms  Contained  Therein..  245 

Exercise  4.  To  Demonstrate  the  Cellulose-decomposing  Power 

of  Aerobic  Organisms  Found  in  the  Soil 246 

Exercise  5.  To  Illustrate  the  Anaerobic  Decomposition  of  Cel- 
lulose by  Soil  and  Fecal  Organisms 248 

Exercise  6.     To  Illustrate  Nitrification  in  Solution 249 

Exercise  7.     To  Illustrate  Denitrification  in  Solution 251 

Exercise  8.  To  Illustrate  the  Non-symbiotic  Fixation  of  Nitrogen 
by  Soil  Organisms  and  Isolation  of  Azotobacter 
Through  Its  Mineral  Food  Requirements 253 

Exercise  9.  A  study  of  the  Symbiotic  Nitrogen-fixing  Organisms 

of  Legumes,  Ps.  Radicicola 256 

Exercise  10.  To  Demonstrate  the  Change  of  Insoluble  Phosphates 
to  a  Soluble  Form  Through  the  Agency  of  Micro- 
organisms  , 263 


X  CONTENTS 

DAIRY  MICROBIOLOGY 

PAGE 

Exercise  1.  A  Comparative  Study  of  the  Number  and  Types  of 

Microorganisms  and  Other  Cells  in  Milk 264 

Exercise  2.  The  Determination  of  the  Bacterial  Content  of 

<  Milk  in  the  Udder 268 

Exercise  3.     To  Illustrate  Extraneous  Contamination 270 

Exercise  4.  To  Investigate  the  Amount  and  Kind  of  Dirt  in 
Milk  and  its  Relation  to  the  Microbial  Content  of 
the  Milk 273 

Exercise  5.  To  Determine  the  Influence  of  Temperature  upon 
the  Keeping  Quality  of  Milk;  Pure  Milk  Com- 
pared with  Market  Milk 278 

Exercise  6.  A  Study  of  the  Pasteurization  of  Milk  or  Cream  by 

Laboratory  Methods 281 

Exercise  7.  Determination  of  the  Number  and  Types  of  Bacteria 

in  Butter 284 

Exercise  8.  To  Determine  the  Number  and  Types  of  Micro- 
organisms in  Cheese 286 

Exercise  9.  A  Comparison  of  the  Bacterial  Content  of  Sweetened 

and  Unsweetened  Condensed  Milks 288 

Exercise  10.  To  Determine  the  Number  and  Types  of ,  Micro- 
organisms in  Cream 289 

PLANT  MICROBIOLOGY 

Exercise  1.     To  Demonstrate  that  Plants  are  Subject  to  Microbial 

Diseases 291 

ANIMAL  DISEASES  AND  IMMUNITY 

Exercise  1.  Animal  Inoculation  in  Bacteriology  for  the  Deter- 
mination of  the  Identity  of  a  Microorganism,  Its 
Pathogenicity  or  Virulence,  or  for  Production  of 

Immunity 295 

Exercise  2.     Isolation  of  Pathogenic  Bacteria  from  Fluids  and 

Tissues  of  Dead  Animals 301 

Exercise  3.     A  Study  of  Bact.  Anthracis 302 

Exercise  4.     The  Preparation  of  Tubuculin 303 

Tuberculin  Test  Chart 306 

Exercise  5.     The  Preparation  of  Black-leg  Vaccine 307 

Exercise  6.     The  Preparation  of  Tetanus  Toxin 308 

Exercise  7.     The  Preparation  of  Tetanus  Antitoxin 309 


CONTENTS  xi 

PAQE 

Exercise  8.    A  Demonstration  of  the  Agglutination  Test 310 

Exercise  9.     A  Study  of  Filterable  Viruses 313 

Exercise  10.     The  Preparation  of  Bacterins  or  Bacterial  Vaccines .  316 
Exercise  11.     To  Demonstrate  Opsonins  and  to  Determine  the 

Opsonic  Index 319 

Exercise  12.     To  Demonstrate  the  Precipitin  Test 321 

Exercise  13.     The  Production  of  a  Hemolytic  Serum 323 

Exercise  14.     To  Demonstrate  the  Complement  Fixation  Test . . .  325 

APPENDIX 

Outline  for  the  Study  of  Microbiology — Special  Media — Table  for 
Identification  of  Bacteria  in  Polluted  Water — Characters  of 
B.  Coli — B.  Typhosus  Groups — Common  Disinfectants — So- 
lutions for  Cleaning  Glassware — Standard  Solutions — Indi- 
cators— Salt  Solutions — Test  Solutions — Mounting  Media — 
Stains — Solutions  for  Use  in  Staining — Steam  Table — Tem- 
perature Conversion  Formulae — Metric  System — List  of  Text 
and  Reference  Books, . ,  .331 


LABORATORY    RULES 


1.  Do  not  bring  coats,   sweaters,   hats,   etc.,  into  the 
laboratory  and  lay  them  on  desks,  etc.;   hang  them  in  the 
place  provided  for  the  purpose. 

2.  Before  beginning  and  after  finishing  work,  the  top 
of  the  desks  must  be  washed  off  with  a  liberal  supply  of 
1-1000   mercuric    chloride.     This   will   destroy   all   micro- 
organisms and  their  spores  and  aid  greatly  in  rendering 
aseptic  technic  possible.     A  large  bottle  of  this  disinfectant 
will  be  found  near  each  desk. 

3.  Do  not  put  string,  paper,  pencils,  pins,  etc.,  in  the 
mouth  nor  moisten  labels  with  the  tongue  while  in  the 
laboratory.     Follow  this  practice  outside  of  the  laboratory 
also.     Food  should  not  be  eaten  in  the  laboratory. 

4.  Observe  all  possible  cleanliness  and  neatness  in  the 
care  of  apparatus,  desk,  microscope,  etc. 

5.  Apparatus  must  be  kept  inside  the  desks,  but  not 
cultures.     Cultures  must  be  kept  at  a  constant  temperature 
in  the  place  fitted  for  this  purpose. 

The  microscope  and  accessories  must  be    returned    to 
the  case  at  the  close  of  the  work. 

6.  Water,  gas,  steam  and  electricity  are  to  be  turned  off 
when  not  in  use.     This  applies  to  the  individual  desks,  large 
sinks,  steam  (including  autoclav),  hot-air  sterilizers,  etc. 

7.  Put  all  solid  waste  material,  cotton,  paper,  matches, 
coagulated  milk,  etc.,  and  waste  liquid  which  will  solidify 
when  cold  (agar,  gelatin)  into  receptacles  provided  for  that 
purpose,  not  into  the  sinks. 

8.  Apparatus,  media,  etc.,  should  be  removed  from  steam 
heaters,  immediately  after  steaming. 

xiii 


xiv  LABORATORY  RULES 

9.  No  cultures  are  to  be  taken  out  of  the  laboratory 
without  the  permission  of  the  head  of  the  department. 

10.  All    accidents,    such    as    spilling   infected    material 
(pathogenic   or  non-pathogenic),    cutting   or   pricking   the 
fingers,  must  be  reported  at  once  to  the  instructor  in  charge. 

Additional  rules  will  be  given  if  necessary,  in  conjunction 
with  special  exercises  or  technic. 

11.  1-1000  mercuric  chloride  will  not  injure  the  skin  if 
not  used  too  often.     Wash  your  hands  in  it  thoroughly  each 
time  before  you  leave  the  laboratory  to  avoid  carrying  away 
undesirable  organisms.     Use  every  precaution  against  in- 
fection. 

12.  At  the  beginning  of  each  laboratory  period  read  over 
carefully  the  directions  for  the  next  exercise  in  order  to 
understand  its  purpose  and  to  make  any  necessary  pre- 
liminary preparations. 

13.  Take  careful  notes  on  all  observations  made  in  the 
study  of  cultures  and  preparations  made  from  them. 


FORM  FOR  WRITING  UP  EXERCISES  IN  THE 
NOTEBOOK 


I.  Object.    A  concise  statement  of  what  the  exercise  is 
intended  to  prove  or  demonstrate  is  to  be  given. 

II.  Apparatus.     This  includes  everything  with  the  excep- 
tion of  the  every-day  tools  such  as  burner,  platinum  needles, 
etc. 

III.  Cultures.     A  brief  morphological  and  cultural  de- 
scription characteristic  of  each  organism  should  be  given, 
also  its  occurrence  and  importance.     Certain  organisms  are 
used  for  a  certain  purpose.     If  this  purpose  is  not  evident, 
ascertain  from  the    references  given  why  these  particular 
organisms  were  used. 

IV.  Method.     State  briefly  but  clearly  your  method  of 
procedure. 

V.  Results.     Give  your  results  in  full.    Tabulate  data  so 
that  they  may  be  comprehended  at  a  glance.    Results  often 
may  be  tabulated  as   +   and   — .     Plot  curves  whenever 
possible. 

VI.  Conclusions.    Draw  the  conclusion  which  your  own 
results  warrant. 

VII.  Error.     You  may  know  that  your  results  and  the 
consequent  conclusions  are  in  error.     If  so,  state  what  you 
consider  to  be  the  correct  results  and  conclusions,  noting 
any  irregularities  or  abnormalities  which  may  have  occurred 
to  change  the  results. 

VIII.  Practical   Application.    Apply  the  principles  in- 
volved in  the  exercise  to  some  practical  purpose. 

XV 


xvi  FORM   FOR  WRITING  UP  EXERCISES 

IX.  References.  Give  the  substance  of  the  references 

placed   at   the  end  of   each  exercise  in  your  own  words 

and    apply   to   the  exercise    in   question.      Do  not  copy 
verbatim. 

Note.     In  writing  up  the  notebook,  details  under  II  and  IV  should 
be  omitted,  only  the  headings  are  necessary. 


PART  I 

GENERAL  MORPHOLOGICAL   AND  CULTURAL 
METHODS 


EXERCISE  1.    CLEANING  GLASSWARE 

Glassware  for  use  in  microbiological  laboratory  work 
should  be  not  merely  clean,  but  chemically  clean.  Test 
tubes,  Petri  dishes,  flasks,  etc.,  are  the  receptacles  used  in 
the  microbiological  laboratory  for  containing  the  different 
nutrient  substances  upon  which  microorganisms  are  to 
subsist.  Very  frequently  free  alkali  may  be  present  on 
new  glassware  in  sufficient  quantity  to  prevent  microbial 
growths  in  the  nutrients  contained  therein.  Prescott  and 
Winslow  in  testing  out  different  glassware  say  that,  "  The 
more  soluble  glassware  yielded  sufficient  alkali  to  the  me- 
dium to  inhibit  four-fifths  of  the  bacteria  present  in  certain 
cases." 

Glassware  which  looks  clean  may  have  been  used  previ- 
ously and  should  be  given  a  thorough  cleaning  to  rid  it  of 
possible  traces  of  mercuric  chloride,  or  other  chemical  having 
germicidal  properties. 

Follow  directions  carefully  and  clean  all  new  and  appar- 
ently clean  glassware  in  the  order  given. 

Cleaning  New  or  Apparently  Clean  Glassware.  All  new 
glassware  should  first  be  treated  with  chromic  acid  cleaning 
solution  (see  appendix  for  all  formula)  before  proceeding 
with  the  directions  for  cleaning  glassware. 

Return  used  cleaning  solution  to  the  glass  receptacle 
provided  for  the  purpose.  Do  not  throw  it  away.  This 

1 


2  GENERAL1  MICROBIOLOGY 

solution  may  'be  Used  Wtii  dxhUzed,  i.e.,  until  dark  green 
in  color. 

Heat  will  facilitate  the  action  of  the  cleaning  solu- 
tion. 

Small  amounts  of  organic  matter  adhering  to  glass- 
ware are  oxidized  by  this  solution,  but  will  not  dis- 
appear until  removed  by  a  suitable  brush  and  cleaning 
powder.  * 

New  Petri  dishes  and  test  tubes  may  conveniently  be 


(a) 


(d) 


o 


FIG.  1. — (a)  Pipette,  (6)  Smith's  Fermentation  Tube,  (c)  Erlenmeyer 
Flask,  (d)  Test  Tube,  (e)  Roux  Tube,  (f)  Petri  Dish,  (g)  Roux 
Flask. 

placed  in  a  large  glass  jar,  covered  with  cleaning  solution 
and  allowed  to  stand  over  night.  Heavy  glass  jars  will 
not  stand  heating  in  steam.  New  flasks  may  be  partially 
filled  with  cleaning  solution  and  placed  in  steam  for  fifteen 
minutes. 

Test  Tubes.     New  test    tubes    should    be   filled  with 
cleaning  solution,  placed  in   a  wire  basket  and  heated  for 

*  Any  inexpensive  fine-grained  cleaning  powder  as  powdered  pum- 
ice stone,  Bon  Ami,  etc.,  may  be  used. 


CLEANING  GLASSWARE  3 

at  least  fifteen  minutes  in  the  steam.  After  removing  test 
tubes  from  the  cleaning  solution: 

1.  Wash  them  in  water  with   a  test-tube   brush,   using 
cleaning  powder  if  necessary. 

2.  Rinse  with  tap  water  till  clean  and  free  from  cleaning 
powder. 

3.  Rinse  with  distilled  water. 

4.  Drain. 

5.  Test  tubes  and  other  glassware,  flasks,  pipettes,  etc., 
may  be  rinsed  with  alcohol  to  facilitate  drying,  then  drained. 

Flasks.     After  treating  flasks  with  cleaning  solution: 

1.  Wash   them   as   clean   as   possible    with   tap   water 
and  a  flask  brush ;  use  cleaning  powder  if  necessary.     (When 
using  cleaning  powder,  empty  all  water  out  of  the  flask, 
wet  the  flask  brush  with  tap  water,  dip  it  in  the  cleaning 
powder  and  then  rub  the  soiled  portions  vigorously.) 

2.  Rinse  with  tap  water  till  clear  and  free  from  cleaning 
powder. 

3.  Rinse  with  distilled  water. 

4.  Drain. 

Petri  Dishes.     After  removing  Petri  dishes  from  the 
cleaning  solution: 

1.  Wash  them  in  water,  using  cleaning  powder  if  nec- 
essary. 

2.  Rinse  with  tap  water.     (It  is  not  necessary  to  use 
alcohol  or  distilled  water.) 

3.  Wipe  immediately  with  a  clean  physician's  cloth. 
Pipettes.     1.  Place   pipettes   delivery   end   down,   in   a 

glass  cylinder  (graduate)  in  cleaning  solution  and  allow 
them  to  stand  over  night.  (Steam  may  break  the  glass 
cylinder) . 

2.  Pipettes  which   have  been  used   should  be  washed 
immediately.     Grease    which    cannot    be    removed    with 
water  should  be  treated  with  10%  NaOH  and  then  with 
cleaning  solution. 

3.  Rinse  with  tap  water,  followed  by  distilled  water. 


4  GENERAL  MICROBIOLOGY 

4.  Rinse  with  alcohol.    (Alcohol  may  be  used  repeatedly.) 

5.  Drain. 

Fermentation  Tubes.     1.  Rinse  with  tap  water. 

2.  Fill  with  cleaning  solution  and  heat  fifteen  minutes 
in  steam  or  allow  to  stand  over  night  if  more  convenient. 

3.  Wash    thoroughly  in    tap  water,  using    a   test-tube 
brush  if  necessary. 

4.  Rinse  in  distilled  water  and  drain. 
Cover-glasses  and  Slides.     1.  Immerse  the  cover-glasses 

or  slides,  one  by  one  in  a  10%  solution  of  sodium  hydrate 
(NaOH)  for  thirty  minutes  only.  This  strength  of  NaOH 
will  etch  the  glassware  if  left  longer. 

2.  Wash  separately  in  tap  water,  handling  with  ordinary 
forceps.* 

3.  Put,  one  at  a  time,  in  cleaning  solution,  and  leave 
over  night  as  convenient. 

4.  Wash  separately  in  water. 

5.  Immerse  in  clean  alcohol  (95%). 

6.  Wipe  with  a  clean  physician's  cloth. 

7.  Store    in   clean   Esmarch    and    deep    culture    dishes 
respectively,  to  keep  free  from  dust. 

Other  Glassware.  Some  modification  of  these  methods 
will  be  adaptable  to  nearly  all  glassware. 

Note  1.  Glassware  containing  liquefiable  solid  media  is  best 
cleaned  by  heating  and  pouring  out  the  material  while  in  liquid 
condition,  then  treating  as  above.  (Solid  media  when  liquefied  by 
heat  should  never  be  thrown  in  the  sink,  as  it  will  solidify  when 
cold  and  clog  up  the  traps  and  drains.) 

Note  2.  Flasks,  test  tubes,  Petri  dishes,  etc.,  containing 
cultures,  must  be  heated  one  hour  in  flowing  steam  before  cleaning. 

Cultures  containing  spores  should  be  autoclaved  previous  to 
cleaning. 

Note  3.  If  cultures  or  media  have  become  dry,  add  water 
before  heating. 

Especial  care  must  be  used  in  cleaning  glassware  in  which  mer- 
curic chloride  or  any  other  disinfectant  has  been  used. 

*  Always  handle  cover-glasses  and  slides  with  forceps. 


STERILIZATION  5 

STERILIZATION 

Sterilization  consists  in  the  destruction  of  all  forms  of 
life.  It  may  be  effected  by  various  agents.  As  applied 
to  the  practical  requirements  of  the  bacteriological  lab- 
oratory many  of  those  agents  such  as  electricity,  sunlight, 
etc.,  are  of  little  value  and  are  limited  in  their  applications; 
others  are  so  well  suited  to  particular  purposes  that  their 
use  is  almost  entirely  restricted  to  such  applications. 

The  Two  General  Methods  of  Sterilization  are: 

A.  Physical. 

1.  Plasmolysis  or  Plasmoptysis. 

2.  Desiccation. 

3.  Heat — (a)  dry  heat;  (6)  moist  heat. 

4.  Light. 

5.  Filtration. 

6.  Dialysis. 

7.  Comminution. 

B.  Chemical. 

1.  Disinfectants,  etc. 

A.     PHYSICAL   AGENTS 

I.  Concentrated   solutions   destroy  microorganisms    by 
withdrawing  water  from  their  cells  (plasmolysis) ,  e.g.,  in  the 
preservation   of   food  by  concentrated  salt  or  sugar  solu- 
tions. 

Microorganisms  accustomed  to  a  concentrated  nutrient 
substrate  may  suffer  plasmoplysis  (bursting  of  the  cell) 
if  placed  in  a  less  concentrated  medium. 

In  either  case,  if  they  are  subjected  gradually  to  the 
changing  conditions,  death  is  delayed  or  prevented. 

II.  Desiccation  is  destructive  to  many  microbes,  espe- 
cially those  which  do  not  form  spores.     For  example,  Ps. 
radicicola  is  very  sensitive  to  desiccation  on   the  ordinary 
cover-glass  or  on  cotton. 


6  GENERAL  MICROBIOLOGY 

III.  Sterilization  by  Dry  Heat. 

1.  Sterilization  in  a  naked  flame. 

2.  Sterilization  in  an  ether  flame. 

3.  Sterilization  in  a  muffle  furnace. 

4.  Sterilization  by  hot  air. 

1.  Sterilization  in  a  Naked    Flame,     (a)  The    simplest 
means  of  sterilizing  a  metal  instrument  is  to  heat  it  to  red- 
ness in  a  flame.     This  method  is  always  adopted  for  ster- 
ilizing platinum,   copper,  etc.,   wires  and  iron  and  nickel 
spatulas,  forceps,  etc. 

A  platinum  needle  should  always  be  carefully  dried 
before  sterilization,  by  holding  it  near  the  flame.  This 
avoids  sputtering,  which  scatters  microorganisms,  especially 
if  moist  material,  e.g.,  fat  or  protein,  on  the  needle  is 
immediately  thrust  into  the  flame. 

(6)  An  instrument  may  be  sterilized  by  flaming  it, 
i.e.,  by  passing  it  rapidly  through  a  hot  flame.  This  method 
is  useful  for  instruments,  etc.,  having  polished  surfaces 
devoid  of  creases  in  which  microorganisms  might  escape 
destruction,  e.g.,  knives,  glass  rods,  handles  of  platinum 
needles,  mouths  of  test  tubes,  flasks,  pipettes,  etc. 

(c)  Deep  wounds  are  sterilized  by  cautery  with  an 
instrument  heated  to  a  dull  red  heat. 

2.  Sterilization  in  an  Ether  Flame.     In  an   emergency, 
small  instruments,  needles,  etc.,  may  be  sterilized  by  dip- 
ping them  in  ether  or  absolute  alcohol  and  after  removal 
lighting  the  adherent  fluid  and  allowing  it  to  burn  off  the 
surface  of  the  instruments.     Repeat  the  process.     It  may 
then  be  safely  assumed  that  the  apparatus  so  treated  is 
sterile. 

3.  Sterilization  in  a   Muffle   Furnace.     Porcelain    filter 
candles  are  sterilized  by  heating  them  to  white  heat  in 
the  muffle  furnace.     This  method   of  sterilization  cannot 
be  applied  to  porcelain  filters  with  metal  fittings,  such  as 
Berkefeld  filters. 

The  destruction  of  autopsied  animals  and  accumulated 


STERILIZATION  7 

wastes  of  the  laboratory  is  also  best  accomplished  in  this 
manner. 

4.  Sterilization  by  Hot  Air.  Exposure  to  hot  air  is  the 
usual  method  of  sterilizing  all  glassware,  instruments  with 
metal  handles,  etc.,  but  it  is  not  suitable  for  organic  sub- 
stances, with  the  exception  of  wool,  cotton  and  paper. 

To  insure  efficient  sterilization,  the  prepared  glassware, 


FIG.  2.— Hot  Air  Sterilizer. 

etc.,  must  be  placed  in  a  gas  or  electrically  heated  oven 
(containing  a  thermometer  registering  over  200°  C.)  whose 
temperature  is  maintained  at  approximately  150°  C.  for 
one  hour,  or  180°  C.  for  ten  minutes.  The  oven  must  be 
allowed  to  cool  down  to  60°  C.  before  opening  the  door 
to  avoid  the  breaking  of  glassware  by  cold-air  currents. 
Cotton,  wool,  and  paper  are  slightly  scorched  at  this  tem- 
perature, 


8  GENERAL  MICROBIOLOGY 

Apparatus  must  be  absolutely  clean  and  dry  before 
being  sterilized. 

IV.  Sterilization  by  Moist  Heat.  Sterilization  by  moist 
heat  may  be  effected  in  one  of  four  ways: 

1.  By    continuous    or    discontinuous    heating    at    low 
temperatures  (56°-80°  C.). 

2.  By   continuous   or   discontinuous   heating   in   water 
at  100°  C. 

3.  By  continuous  or  discontinuous  heating  in  flowing 
steam  at  100°  C. 

4.  By  one  heating  in  superheated  steam  (steam  under 
pressure)  at  temperatures  above  100°  C.,  generally  115°  C. 
(about  10  Ibs.  pressure)  or  120°  C.  (about  15  Ibs.). 

1.  Sterilization  by  Continuous  or  Discontinuous  Heating  at 
Low  Temperatures.     Some  substances  used  as  culture  media, 
being   rich   in   volatile   or   otherwise    chemically   unstable 
substances,  cannot  be  heated  to  100°  C.  without  a  marked 
alteration  (e.g.,  coagulation)  and  to  some  extent  a  destruc- 
tion of  their  properties;  blood  serum,  for  example. 

Pasteur  showed  that  such  media  can  be  better  ster- 
ilized by  heating  them  at  a  low  temperature  (55°-60°  C.) 
for  a  long  time  than  at  a  high  temperature  (70°  C.  or 
even  100°  C.)  for  a  short  time.  In  this  process,  heat  is 
not  applied 'directly,  as  a  rule.  Control  of  the  temperature 
is  ordinarily  accomplished  by  means  of  water  heated  to 
the  degree  desired. 

Prolonged  heating  at  a  low  temperature  constitutes 
pasteurization.  In  practice,  however,  it  is  found  that  in 
order  to  kill  all  organisms  pasteurization  must  be  com- 
bined with  the  method  of  discontinuous  heating  devised 
by  Tyndall.  Albuminous  media  subjected  to  the  Tyndall 
method  must  be  incubated  finally  at  37°  C.  for  forty-eight 
hours  to  eliminate  all  specimens  showing  contamination. 

2.  Sterilization  by  Continuous  or  Discontinuous  Heating 
in  Water  at  100°  C.     (a)  Continuous   Heating.     Water  at 
100°  C.  destroys  the  vegetative  forms  of  bacteria  almost 


STERILIZATION 


9 


instantaneously,  and  spores  in  from  five  to  fifteen  minutes 
ordinarily,  although  many  spores  of  resistant  species  are  not 
killed  by  several  hours'  heating  at  100°  C.  Water  suspected 
of  sewage  contamination  may  thus  be  rendered  safe  for 
drinking  purposes  simply  by  boiling  for  a  few  minutes. 

This  method  is  applicable  to  metal  instruments,  syringes, 
rubber  stoppers,  rubber  and  glass  tubing,  and  other  small 
apparatus. 

(b)  Discontinuous  Heating.  (Tyndall  method.)  Tyndall 
observed  that  certain  resistant 
forms  found  in  an  infusion  made 
from  hay  were  not  destroyed  by 
heating  the  infusion  at  100°  C., 
once,  even  when  the  temperature 
was  sustained  for  a  prolonged 
period,  yet  by  boiling  it  for  a 
short  time  on  three  successive 
days  all  living  organisms  were 
destroyed.  His  theory  was  that 
by  heating  at  100°  C.,  the  vege- 
tative forms  but  not  the  spores 
were  killed.  The  latter  germinate 
as  the  fluid  cools  and  are  killed 
during  the  second  heating.  A 
few  spores,  however,  escape  de- 
struction at  the  second  heating; 

these  will  have  germinated  by  the  time  the  third  heating  is 
due.  After  the  third  heating  sterilization  is  accomplished. 

The  explanation  now  given,  however,  is  that  the  resist- 
ance of  microorganisms  is  gradually  lowered  under  the 
influence  of  repeated  heatings.  This  principle  of  heating 
on  three  successive  days,  a  medium  to  be  sterilized  is  now 
known  as  the  Tyndall  method  of  sterilization.  In  general 
laboratory  practice,  steam  is  used  instead  of  water  at  100° 
C.,  but  this  necessitates  special  apparatus,  whereas  water 
lends  itself  readily  to  the  means  at  hand. 


FIG.  3.— Arnold  Steam 
Sterilizer. 


10  GENERAL  MICROBIOLOGY 

The  physical  nature  of  the  medium,  the  extraordinary 
resistance  of  the  spores  of  certain  species  of  bacteria  or 
both  in  combination,  may  require  that  this  intermittent 
heating  be  carried  on  over  a  longer  period  of  time,  i.e., 
four,  five,  six,  etc.,  days  in  succession  for  the  same  or  a 
longer  period  each  time,  or  that  the  period  between  inter- 
mittent heatings  be  lengthened  from  twenty-four  hours 
to  forty-eight  hours. 

Tyndall's  method  is  valuable  in  that  media  of  delicate 
composition  may  be  sterilized  without  producing  undesirable 
changes,  such  as  are  often  produced  by  the  high  tempera- 
ture of  the  autoclav. 

3.  Sterilization  in  Flowing  Steam  at  100°  C.     Continuous 
or  Discontinuous,     (a)  Continuous  Heating.    Simple  boiling 
or  exposure  to  steam  at  100°  C.,  even  though  the  exposure 
be  prolonged,  is  not  a  reliable  method  of  sterilization.    When 
microorganisms  have  been  dried,   their  resistance  to  the 
effects  of  heat  is  much  enhanced,  and  especially  is  this  the 
case  when  they  are  mixed  with  substances  of  a  colloidal 
nature.     Certain  resistant  forms  of  protoplasm  known  as 
spores  may  not  be  destroyed  by  one  heating  to  100°  C., 
even  when  the  temperature  has  been  maintained  for  sev- 
eral minutes. 

(6)  Discontinuous  Heating.  General  use  for  the  ster- 
ilization of  media. 

This  principle  of  sterilization  advanced  by  Tyndall 
finds  its  widest  application  in  bacteriological  work  with 
the  use  of  flowing  steam.  High-pressure  steam  may  be 
utilized  to  good  advantage  if  a  central  heating  station  is 
available.  The  Arnold  sterilizer  makes  use  of  steam  for 
the  sterilization  process  and  lends  itself  readily  to  both  the 
continuous  and  discontinuous  method. 

4.  Sterilization    by    Superheated  Steam    (under  pressure 
and  therefore   above  100°  C.).     Water,  syringes,  surgical 
dressings,     bedding,    india-rubber    apparatus,    filters,    old 
cultivations,  culture  media,  etc.,  not  injured  by  high  tern- 


STERILIZATION 


11 


peratures,  may  be  more  quickly  sterilized  by  heating  in 
steam  under  pressure. 

Exposure  to  steam  at  a  temperature  of  115°  C.  for  twenty 
minutes  is  in  most  cases  sufficient  to  insure  sterilization, 


FIG.  4. — Autoclav,  Horizontal, 
for  Steam  or  Gas. 


FIG.  5. — Autoclav,  Vertical, 
for  Gas  Only. 


but  some  media,  potato  for  instance,  require  a  temperature 
of  120°  C.  for  ten  to  fifteen  minutes.  It  is  now  realized 
that  media  subjected  to  this  high  temperature  undergo 
hydrolytic  changes  which  render  them  unsuitable  for  the 
cultivation  of  more  delicate  microorganisms.  Sterilization 


12  GENERAL  MICROBIOLOGY 

in  the  superheated  steam  is  carried  on  in  a  special  apparatus 
called  an  autoclav,  which  may  be  so  constructed  as  to  run 
by  direct  or  indirect  steam.  The  latter  is  the  more  desir- 
able for  the  sterilization  of  media. 

V.  Sterilization  by  Light.     Light  seems  to  act  by  pro- 
ducing powerful  chemical  germicides,  probably  organic  per- 
oxides, in  the  medium  surrounding  the  bacteria.     Certain 
rays  of  light,  the  blue,  violet  and  ultraviolet  in  particular 
are  destructive  to  living  cells.    It  is  to  these  rays  that  sun- 
light owes  its  disinfecting  action.     Practical  use  has  been 
made   of   the   ultraviolet    rays   in   water    sterilization    by 
employing  the  Cooper-Hewitt  mercury  vapor  lamp  having 
a  quartz  instead  of  a  glass  tube,  as  these  rays  do  not  pass 
through  glass. 

VI.  Sterilization    by    Filtration.     Sterilization  may  be 
effected    by    the    filtration    of    gases    or    liquids    through 
materials  which  will  retain  microorganisms. 

The  best  example  of  the  filtration  of  gases  is  the  use 
of  cotton  plugs  in  flasks  and  tubes  containing  microorgan- 
isms. The  cotton  is  porous  enough  to  allow  the  necessary 
interchange  of  gases  but  will  allow  neither  dust  nor  foreign 
microorganisms  to  enter.  The  sterilization  of  air  or  other 
gases  if  fore  3d  through  cotton  would  depend  upon  the 
thickness  of  the  cotton  layer  and  also  upon  the  force  which 
was  exerted. 

Certain  fluids  used  in  bacteriological  work  cannot  be 
subjected  even  to  a  moderate  amount  of  heat  without  pro- 
foundly altering  their  nature.  In  order  to  make  such  a 
fluid  sterile,  it  is  passed  through  a  cylindrical  vessel,  closed 
at  one  end  like  a  test  tube,  and  made  either  of  porous 
"  biscuit"  porcelain,  hard  burnt  and  unglazed  (Chamber- 
land  filter)  or  of  kieselguhr,  a  fine  diatomaceous  earth 
(Berkefeld  filter)  and  termed  a  bougie  or  a  candle. 

The  pores  of  the  finer  filters  are  so  small  that  while 
liquids,  and  solids  in  solution  pass  through,  microorganisms 
are  retained  and  the  liquid  passes  through  in  a  germ-free 


STERILIZATION  13 

condition.  Pasteur  in  his  early  work  utilized  plaster  plates 
as  the  filtering  medium,  but  as  a  result  of  Chamberland's 
researches,  porous  porcelain  now  supersedes  plaster.  Finely 
shredded  asbestos  packed  tightly  in  a  Gooch  crucible  will 
serve  as  a  bacterial  filter  provided  the  layer  of  asbestos 
is  sufficiently  thick.  The  rate  of  filtration  is  usually  very 
slow  because  the  pores  of  the  filter  are  so  very  minute; 
therefore  to  overcome  this  disadvantage  either  aspiration 
or  pressure  is  generally  employed  to  hasten  the  process. 
This  method  may  not  exclude  filterable  organisms. 

VII.  Sterilization  by    Dialysis.     In   one    of    the   more 
recent  methods  devised  for  the  preparation  of  antirabic 
vaccines  the  vaccine  is  prepared  by  placing  the  virus  (spinal 
cord  of  a  rabid  rabbit)  in  a  collodion  sac  and  dialyzing  it 
in  running  distilled  water.     The  living  virus  is  destroyed, 
yet   its   immunizing    properties    are    retained   unimpaired. 
Quite  the  opposite  effect  may  be  obtained  under  some- 
what different  circumstances.     If  a  collodion  sac  containing 
a  suspension  of  a  pathogenic  organism  be  placed  in  the 
body  cavity  of  a  susceptible  animal  the  organisms  within 
the  sac  thrive,  being  nourished  by  the  body  fluids  which 
diffuse  through  the  semi-permeable  membrane. 

GUMMING,  J.  G.:  Rabies — Hydrophobia.  A  study  of  fixed  virus, 
determination  of  the  M.  L.  D.,  vaccine  treatment  (Hogyes, 
Pasteur,  and  dialyzed  vaccine),  and  immunity  tests.  Journal 
of  Infectious  Diseases,  Vol.  XIV  (1914),  pp.  33-52. 

VIII.  Comminution  or  the  actual  crushing  of  the  micro- 
bial    cells  is  resorted  to    for    demonstrating    intracellular 
enzymes. 


14  GENERAL  MICROBIOLOGY 


B.     CHEMICAL  AGENTS 

I.  Sterilization  by  Disinfectants.  Sterilization  by  dis- 
infectants has  but  limited  use  in  bacteriological  work. 
The  amount  of  disinfectant  necessary  to  destroy  existing 
organisms  in  a  nutrient  medium  is  greater  than  the  amount 
necessary  to  inhibit  multiplication  of  an  organism  which 
may  subsequently  be  used  as  an  inoculum;  the  medium 
is  therefore  rendered  useless. 

1.  Disinfectants  may  be  used  for  any  apparatus  which 
will  not  come  in  direct  contact  with  culture  media  or  with 
the  organisms  under  investigation.     Fixed  non-volatile  dis- 
infectants must  be  employed,  since  the  vapors  given  off  by 
volatile   compounds   hinder   the   growth   of   organisms   on 
culture  media. 

2.  Disinfectants  are  in  general   use   for  sterilizing  the 
hands,  woodwork,  for  washing  out  vessels  and  sterilizing 
instruments  during  inoculation  and  other  experiments. 

As  an  example,  1-1000  mercuric  chloride,  1.5%  formalin, 
5%  phenol,  2%  compound  solution  of  cresol,  etc.,  are  cheap 
and  adaptable  in  many  cases.  Tincture  of  iodin  is  valuable 
for  painting  wounds. 

The  common  soaps,  and  more  particularly  green  soap, 
have  a  plight  germicidal  value,  and  this  in  conjunction 
with  their  solvent  action  upon  fats  and  protein,  and  the 
mechanical  cleansing  which  accompanies  their  use,  justifies 
assigning  them  an  important  place  among  the  chemical 
disinfectants. 

Disinfectants  used  for  sterilizing  the  skin  before  col- 
lecting pus,  blood,  etc.,  from  the  living  subject  must  be 
carefully  removed  by  washing  the  part  well  with  alcohol 
before  collecting  material,  otherwise  the  presence  of  the 
disinfectant  would  materially  interfere  with  the  subsequent 
growth  of  organisms  in  the  culture. 

3.  Disinfectants  are  also  added  to  sterile  filtrates  which 
are  no  longer  required  as  culture  media.     For  this  pur- 


GLASSWARE  FOR  STERILIZATION  15 

pose  a  small  quantity  of  some  disinfectant  (such  as  thy- 
mol or  camphor)  which  is  without  chemical  action  on  the 
constituents  of  the  fluid  is  selected. 

An  amount  of  carbolic  acid  (0.5%)  or  other  chemical 
is  frequently  added  to  vaccines,  bac terms,  serums,  etc.,  for 
preservative  purposes. 

4.  Disinfectants  are  sometimes  used  to  sterilize  a  culture 
when  the  products  of  the  microorgansims  are  under  inves- 
tigation. Chloroform,  ether,  toluol,  oil  of  garlic  or  mustard, 
etc.,  which  may  be  driven  off  afterward  by  evaporation, 
are  among  the  most  useful  in  this  connection. 

II.  Sterilization  by  Antiseptics.  Chemical  reagents  such 
as  belong  to  the  class  known  as  antiseptics,  i.e.,  substances 
which  inhibit  the  growth  of,  but  do  not  destroy  bacterial 
life,  are  obviously  useless. 

REFERENCES 

EYRE:     Bacteriological  Technic.     Second  Edition  (1913),  pp.  26-48. 
BESSON:     Practical  Bacteriology,  Microbiology,  and   Serum  Therapy 

(1913),  pp.  3-27. 

MARSHALL:     Microbiology  (1911),  pp.  64-67. 
EULER:     General  Chemistry  of  the  Enzymes  (1912),  pp.  118-123.. 

EXERCISE    2.     PREPARATION     OF    GLASSWARE    FOR 
STERILIZATION 

The  mouths  of  test  tubes,  fermentation  tubes,  pipettes, 
etc.,  are  ordinarily  plugged  with  cotton  before  sterilization. 
For  this  purpose  cotton  is  ideal  as  it  is  cheap  and  adaptable, 
serves  to  filter  out  microorganisms  from  the  air,  while  allow- 
ing the  ready  diffusion  of  gases,  and  after  once  used  it  may- 
be  burned. 

Paper  (ordinary  newspaper)  may  be  used  to  wrap  glass- 
ware as  Petri  dishes,  deep-culture  dishes,  pipettes,  etc.,  which 
one  wishes  to  store  in  a  sterile  condition  and  for  which 
cotton  is  not  adaptable. 

Glassware  is  sterilized  for  the  purpose  of  destroying 


16  GENERAL  MICROBIOLOGY 

microorganisms  present  on  its  surface  and  in  or  on  the  cotton 
or  paper  used  respectively  for  plugging  or  wrapping.  After 
sterilization  the  cotton  and  paper  serve  to  prevent  micro- 
organisms from  entering  and  contaminating  the  sterile 
utensils. 

Dry  heat,  though  not  as  effective  a  germ  destroyer  as 
moist  heat,  is  more  adaptable  to  the  sterilization  of  empty 
culture  flasks,  pipettes  and  other  glassware.  Hot-air  steril- 
ization not  only  accomplishes  the  sterilization  of  the  glass- 
ware, cotton  plugs,  etc.,  but  "  sets  "  the  plugs  so  that  they 
may  be  handled  with  greater  facility. 

All  glassware  must  be  absolutely  clean  and  dry  or  contain 
traces  of  alcohol  only  before  preparing  for  sterilization; 
otherwise  sterilization  cannot  be  accomplished.  If  consider- 
able moisture  is  present  in  test  tubes,  flasks,  etc.,  it  will  not 
evaporate  during  the  hot-air  sterilization  process,  and  it  is 
very  evident  that  the  temperature  of  such  moist  portions 
of  the  glassware  will  not  reach  or  at  least-  will  not  exceed 
100°  C. 

Directions.  Test  tubes  and  flasks  are  plugged  with 
cotton.  The  ordinary  forceps  are  used  for  this  purpose. 
(A 'glass  rod  may  also  be  used.)  A  small  piece  of  cotton  is 
grasped  on  the  edge  with  the  forceps  and  inserted  in  the 
mouth  of  the  test  tube.  Plugs  should  project  into  test 
tubes  from  3  to  4  cms.,  and  from  3  to  5  cms.  into  the 
neck  of  flasks,  according  to  the  size  of  the  flask.  Only  an 
amount  of  cotton  should  project  out  of  the  mouth  that  is 
sufficient  to  protect  the  outward  turned  portion  (lip)  of  the 
test  tubes  or  flasks  from  dust.  A  "  Christmas-tree  "  effect 
is  to  be  avoided.  Plugs  should  not  be  so  tight  as  to  be 
removed  with  difficulty,  nor  so  loose  as  to  offer  no  resistance 
to  removal.  A  little  experience  will  suffice  to  demonstrate 
the  amount  of  cotton  to  use  and  the  firmness  with  which 
the  plug  should  fit. 

Cotton  plugs  for  test  tubes,  flasks,  etc.,  may  be  rolled. 
This  kind  of  plug  is  more  stable  and  may  be  used  several 


GLASSWARE  FOR  STERILIZATION 


17 


times.     Have  the  instructor  demonstrate  the  method  of 
rolling. 

For  hot-air  sterilization,  test  tubes  plugged  with  cotton 
may  be  tied  in  large  bundles  or  placed  in  wire    baskets 


FIG.  6.— (a)  Proper  Plug:  (6)  Plug 
too  Shallow  and  too  Loose,  too 
Much  Projecting;  (c)  Plug  too 
Loose,  too  Little  Projecting. 


FIG.  7. — Copper  Cyl- 
inder for  Sterilizing 
Pipettes. 


(never  in  agate  cups),  cotton  plugs  up.  A  few  test  tubes 
should  not  be  placed  in  a  large  wire-basket  or  in  a  wire 
test-tube  rack,  as  it  is  necessary  to  economize  space  in  the 
hot-air  sterilizer. 

Petri  dishes  are  wrapped  separately  in  paper  and  tied 
together  in  sets  of  three,    One  sheet  of  newspaper  makes 


18  GENERAL  MICROBIOLOGY 

four  papers  of  proper  size  for  wrapping  Petri  dishes  and  is 
inexpensive. 

Three  or  more  Petri  dishes  may  be  wrapped  together 
if  all  are  to  be  used  at  the  same  time.  Mark  each  plainly 
with  the  desk  number. 

Pipettes.  Place  a  piece  of  cotton  in  the  bottom  of  a  test 
tube,  plug  the  top  only  of  the  pipette  with  cotton  (not  too 
tightly},  leaving  but  little  of  the  cotton  projecting  out. 
Wrap  a  small  portion  of  cotton  around  the  lower  third  of 
the  pipette,  insert  the  pipette  into  the  test  tube  until  the 
tip  rests  on  the  cotton,  making  the  cotton  wrapping  serve 
as  a  plug  for  the  tube. 

Wrap  pipettes  so  prepared  in  paper  (one  layer)  and  tie 
and  mark  them  plainly  with  the  desk  number. 

A  covered  metal  case  is  often  used  for  holding  pipettes 
to  be  sterilized.  The  upper  end  of  the  pipettes  are  plugged 
with  cotton,  the  pipette  inserted  in  the  case,  the  open  end 
of  the  case  plugged  with  cotton,  and  the  cover  replaced. 
(This  latter  method  is  not  recommended  for  the  new  student, 
as  the  necessity  of  careful  technic  in  removing  a  sterile 
pipette  from  the  case  without  contaminating  those  remaining 
is  difficult  to  impress  upon  him) . 

Fermentation  tubes  are  plugged  with  cotton  as  directed 
for  test  tubes;  the  cotton  plug  should  not  project  into  the 
bulb. 

Deep  culture  dishes  are  wrapped  singly  in  paper  as 
directed  for  Petri  dishes. 

Slides  and  cover-glasses  are  generally  sterilized  by 
flaming,  but  only  as  needed. 

NUTRIENT   MEDIA 

"  Chemically,  like  all  other  living  cells,  microorganisms 
consist  of  organic  and  inorganic  nitrogen  and  mineral  salts; 
it  is  therefore  necessary  in  order  to  grow  a  microorganism, 
that  these  three  classes  of  substances  be  made  available, 
together  with  oxygen,  which  is  an  essential  to  the  life  of  all 


NUTRIENT  MEDIA  19 

living  structures.  Finally  a  certain  amount  of  moisture 
is  absolutely  necessary."  (Besson.) 

A  food  prepared  for  the  growth  of  microorganisms  is 
given  the  general  term  nutrient  medium.  A  large  number  of 
microorganisms  will  grow  readily  in  or  upon  easily  available 
nutrient  media,  as  milk,  bouillon,  etc.  Some  microorgan- 
isms have  widely  differing  food  requirements  and  need  for 
growth  nutrient  media  differing  widely  in  their  composition. 

However,  there  are  a  few  general  rules  that  must  be 
applied  in  the  preparation  of  all  nutrient  media  for  the  use 
of  microorganisms.  These  are  briefly,  that:  Every  culture 
medium  must — 1.  Contain  substances  necessary  for  giowth. 
2.  Be  of  suitable  reaction.  3.  Be  contained  in  vessels 
which  afford  protection  from  contamination  from  without. 

Classification  of  Nutrient  Media.  Culture  media  may 
be  classified  as: 

I.  Natural  Media — as    occurring  in  nature,  e.g.,  milk, 
potato  and  other  vegetables,  meat  and  meat  products,  blood 
and  blood  serum,  egg,  soil,  etc. 

II.  Prepared  media,  i.e.,  made  in  the  laboratory.     These 
are: 

(a)  Of  unknown  chemical  composition;  e.g.,  nutrient 
agar,  gelatin,  etc. 

(6)  Synthetic;  i.e.,  chemical  composition  known,  e.g., 
Giltay  solution  for  denitrifying  organisms. 

Or  as: 

I.  Liquid  Media.     These  include: 

A.  Media  made  from    animal    tissue    and  fluids,  e.g., 
nutrient  broth,   serum  broth,  carbohydrate   broths,   milk, 
blood,  nitrate  peptone  solution,  Dunham's  solution. 

B.  Media  made  from  vegetable  tissue.     Among  these  are: 
Malt  extract  (germinated  barley),  beer  wort,  yeast  extract, 
hay  infusion,  natural  fruit  juices,  wines   (fermented   fruit 
juices). 

C.  Synthetic  media. 

II.  Solid  Media.    These  mav  be  classified  as: 


20  GENEKAL  MICROBIOLOGY 

A.  Liquefiable,  e.g.,  nutrient  agar,  nutrient  gelatin. 

B.  Non-liquefiable,    including:     1.  Media    liquid    in    a 
natural  state  but  which,  once  solidified,  cannot  be  liquefied 
by  physical  means,  e.g.,  media  prepared  from  albuminous 
fluids  and  tissues  such  as  egg,  blood  serum,  etc.,  or  synthetic 
media  solidified  with  sodium  silicate. 

2.  Media  which  are  solid  in  the  natural  state,  e.g., 
vegetable  media  such  as  potato,  carrot,  banana,  etc. 

EXERCISE  3.     TITRATION  OF  MEDIA 

The  titration  of  bacteriological  media  made  from  meat 
is  an  important  step  in  their  preparation,  as  microorganisms 
are  sensitive  to  the  reaction  of  the  nutrient  substrate. 

Procedure.  The  following  method  is  used  for  laboratory 
media,  with  the  exception  of  milk,  wort,  cider,  vinegar,  fruit 
juices,  etc.  See  p.  22. 

1.  Put  5  c.c.  of  the  medium  to  be  tested  and  45  c.c.  of 
distilled  water  in  an  evaporating  dish. 

2.  Boil  briskly  one  minute  with  constant  stirring   (to 
drive  off  all  dissolved  CO2  which  registers  as  acidity). 

$.  Add  1  c.c.  phenolphthalein  solution  for  indicator. 

4.  Titrate  while  hot,  preferably  while  boiling,  with  N/20 
sodium  hydroxide,  or  N/20  hydrochloric  acid  as  the  case  de- 
mands. '  A  faint  but  distinct  permanent  rose  color  marks  the 
end  point.    This  color  should  remain  permanent  for  five  minutes. 

5.  Compute  and  record  the  reaction  of  the  medium  in 
degrees  of  Fuller's  scale,  which  is  the  number  of  cubic  centi- 
meters of  normal*   acid    or  alkali    present   in    1000  cubic 

*  A  solution  is  said  to  be  normal  when  it  contains  1  gram  equiv- 
alent of  an  acid  or  base  in  1  liter. 

A  gram  equivalent  of  an  acid  or  a  base  is  that  quantity  which  is 
equivalent  to  or  will  neutralize  1  gram  molecule  of  a  mono-basic  acid 
or  of  a  mon-acid  base. 

The  advantage  of  the  system  is  that  1  c.c.  of  any  normal  solution 
will  exactly  neutralize  or  be  exactly  equivalent  to  1  milligram  equiva- 
lent of  any  acid  or  base.  (Noyes,  Wm.  A.,  Textbook  of  Chemistry, 
1913,  p.  184.) 


TITRATION  OF  MEDIA  21 

centimeters  of  the  medium,  using  phenolphthalein  as  indi- 
cator. 

6.  Alkaline  media  are  denoted  by  placing  a  minus  (  — ) 
sign  before  the  number  of  degrees  of  alkalinity;  thus,  —15° 
would  indicate  that  the  medium  was  15°  alkaline,  or  that 
15  c.c.  normal  acid  must  be  added  per  liter  to  neutral- 
ize it. 

Acid  media  are  denoted  by  placing  a  plus.  (+)  sign 
before  the  number  of  degrees  of  acidity;  thus,  +15° 
would  indicate  that  the  medium  was  15°  acid  or  that 
15  c.c.  of  normal  alkali  must  be  added  per  liter  to  neu- 
tralize it. 

Example. 

Burette  reading  after  titrating 5.4  c.c. 

Burette  reading  before  titrating 2.0  c.c. 

Number  of  c.c.  N/20  NaOH  required  

to  neutralize  the  acid  in  5  c.c.  of 

the  medium §.4  c.c. 

If  5  c.c.  of  the  medium  (which  is  1/20  of  100  c.c.)  require 
3.4  c.c.  of  1/20  normal  NaOH  to  neutralize  the  acid  pres- 
ent, 100  c.c.  of  the  medium  would  require  20X3.4  c.c.  or 
68  c.c.  of  1/20  normal  NaOH. 

As  a  normal  solution  is  20  times  the  strength  of  a  1/20 
normal  solution,  100  c.c.  of  the  medium  would  require  1/20 
of  68  c.c.  or  3.4  c.c.  of  normal  NaOH  for  neutralization;  and 
one  liter  or  1000  c.c.  of  medium  would  require  10X3.4 
c.c.  or  34  c.c.  N/l  NaOH  for  neutralization;  i.e.,  the 
medium  is  34°  acid.  Fuller's  scale.  This  is  the  litre  of  the 
medium. 

When  N/20  acid  or  alkali  and  a  5  c.c.  portion  of  medium 
(in  45  c.c.  of  distilled  water)  are  used,  each  1/10  of  1  c.c. 
corresponds  to  1°  Fuller's  scale. 

Adjustment  of  Reaction.  If  it  is  desired  to  leave  the 
medium  with  a,  e.g.,  +15°  reaction,  we  have: 


22  GENERAL  MICROBIOLOGY 

Acidity  of  the  medium 

(+34°) 3.4  c.c.  per  100  c.c.  of  the  medium 

Desired  acidity  (+15°) . .  1.5  c.c.  per  100  c.c.  of  the  medium 
Amount  of  normal  alkali  - 

to  be  added 1.9  c.c.  per  100  c.c.  of  the  medium 

or  10X1.9  c.c.  =  19  c.c.  N/l  NaOH  per  1000  c.c.  of  medium 

Since  normal  solutions  are  of  equal  strength  by  volume, 
that  is,  1  c.c.  of  N/l  acid  will  just  neutralize  1  c.c.  of  N/l 
alkali,  it  will  readily  be  seen  that  if  15  c.c.  N/l  NaOH  are 
required  to  neutralize  the  acid  present  in  1  liter  of  medium, 
then  there  must  be  present  in  that  liter  exactly  15  c.c.  of 
N/l  acid,  or  we  should  say  the  reaction  is  (  +  15°)  fifteen 
degrees  acid.  For  any  other  degree  of  acidity  add  enough 
normal  alkali  to  reduce  the  acidity  to  the  point  desired. 

The  reaction  of  a  medium  changes  somewhat  after  its 
neutralization,  especially  during  sterilization,  but  also  upon 
standing  afterward  at  ordinary  temperature.  -  This  change 
is  toward  an  increased  acidity,  and  is  most  marked  in 
media  rich  in  dextrose.  Consequently  it  is  necessary  to 
determine  the  titre  of  a  medium  at  the  time  it  is  used  rather 
than  to  quote  figures  obtained  before  sterilization. 

MILK,  CIDER,  VINEGAR,  WORT,  AND  FRUIT  JUICES 

Procedure.  1.  Into  an  evaporating  dish  measure  5  c.c. 
of  the  medium  to  be  tested,  by  means  of  suitable  pipette. 
Make  up  to  50  c.c.  with  distilled  water. 

Do  not  heat.  The  above  media  should  not  be  heated 
before  titration,  as  they  contain  volatile  acids  or  other  organic 
substances  which  may  register  as  acid  and  which  may  be 
driven  off  by  boiling, 

2.  Add  1  c.c.  phenolphthalein  solution. 

3.  Add,    gradually,    from    an    accurate    burette,    IN/20 
NaOH  until  the  first  permanent  pink  appears. 

4.  Note  the  amount  of  NaOH  required  for  the  titration. 

5.  Always  run  duplicates. 


MILK  23 

6.  Record  as  degrees  of  acidity  the  number  of  c.c.  of 
N/l  NaOH  which  would  be  required  to  neutralize  one  liter 
of  medium. 

MILK 

Milk  is  valuable  as  a  nutrient  medium  for  microorganisms 
because:  It  is  a  natural  nutriment  and  almost  ideal  for  a 
large  number  of  microorganisms.  Its  composition,  averag- 
ing 3.40%  fat,  3.50%  casein  and  albumen,  4.50%  milk 
sugar,  0.75%  ash,  87.75%  water,  is  an  evidence  that  it 
furnishes  food  in  an  excellent  form  for  most  microorganisms. 

The  biochemical  activities  of  many  bacteria  reveal  them- 
selves definitely  in  the  changes  which  milk,  especially  litmus 
milk,  undergoes.  Many  of  these  changes  are  seen  macro- 
scopically.  Some  of  these  are: 

(a)  Acid  Production.  The  lactose,  C^IfeOn  (milk 
sugar),  is  first  inverted,  forming  two  hexose  molecules,  1  mol. 
dextrose  and  1  mol.  galactose. 


And  each  molecule  of  hexose  yields  two  molecules  of 
lactic  acid: 

hexose  =  lactic  acid. 

C6H1206  =  2CH3CH(OH)COOH. 

The  blue  litmus  is  turned  red. 

(b)  AlKali  Production.     Litmus  becomes   darker  blue. 
This  change  very  often  accompanies  peptonization. 

(c)  Reduction  (Decolorization  of  Litmus).     This  is  due 
to  the  reduction  of  the  coloring  matter  (litmus).     Many 
microorganisms  secrete  enzymes  which  produce  hydrogen. 
The  hydrogen  combines  with  the  litmus,  reducing  it  to  its 
leuco-compound  (colorless)  .     (Methylen  blue  becomes  color- 
less under  like  conditions.)     That  this  is  a  reduction  and 
not  a  destruction  may  be  demonstrated  by  shaking  the 
decolorized  culture  with  a  few  cubic  centimeters  of  hydro- 
gen peroxid.     The  bacteria  which  decolorize  the  litmus  also 


24  GENERAL  MICROBIOLOGY 

reduce  the  hydrogen  peroxid  to  E^O  and  nascent  oxygen 
which  reoxidizes  the  reduced  litmus  (showing  by  the  reac- 
tion of  the  milk  the  type  of  microorganisms  present).  Re- 
oxidation  takes  place  slowly  under  natural  conditions. 
Reduction  may  take  place  when  milk  is  acid,  alkaline  or 
neutral. 

(d)  Curdling  through  Acid  Production.     The  casein,  like 
most  proteins,  is  amphoteric,  i.e.,  it  is  capable  of  reacting  both 
as  a  weak  acid  and  a  weak  base.     The  otherwise  insoluble 
casein  is  found  to  be  in  the  milk  in  a  partially  dissolved 
state  (colloidal),  due  to  its  combination  with  the  calcium 
salts:    the  calcium  that  was  formerly  combined  with  the 
casein,  through  the  production  of  acid  by  certain  micro- 
organisms, now  combines  with  the  lactic  acid;    as  a  result 
the  casein  precipitates,  causing  curdling  (coagulation).     Lit- 
mus is  turned  decidedly  red.     Milk  having  an  acid  curd 
will  titrate  above  +50°. 

(e)  Rennet  Curd.    Coagulation  may  also  take  place  when 
the  medium  is  neutral  or  only  slightly^  acid.     This  pro- 
duction of  curd  is  due  to  a  rennet-like  enzyme  produced  by 
microorganisms,  and  is  similar  to  the  action  of  the  rennet 
used  to  curdle  milk  in  cheese  factories. 

Many  spore-forming  species  are  found  under  the  group 
of  rennet-producing  organisms.  Rennet  curd  is  usually 
followed  by  peptonization. 

(/)  Peptonization.  The  curd  produced  by  acid  or  ren- 
net-forming microorganisms  may  gradually  disappear,  leav- 
ing only  a  whey-like  liquid.  This  is  caused  by  certain 
bacteria  which  produce  proteolytic  enzymes  that  digest  the 
curd  and  render  it  soluble.  This  liquefaction  of  solid  pro- 
teins like  gelatin,  fibrin,  boiled  egg  white,  milk  curd,  etc.,  is 
due  to  two  groups  of  enzymes,  pepsin  and  trypsin. 

The  pepsin  of  the  animal  body  acts  only  in  an  acid 
medium  (present  in  the  stomach). 

The  trypsin  of  the  animal  body  acts  only  in  alkaline 
medium  (present  in  the  intestine). 


PREPARATION  OF  LITMUS  MILK  25 

The  pepsin-  and  trypsin-like  enzymes  produced  by  micro- 
organisms cannot  be  thus  separated  by  their  activity  in  a 
medium  of  certain  reaction;  this  varies  with  the  species  of 
microorganism  and  with  environmental  conditions.  Pep- 
tonization  of  milk  usually  takes  place  in  a  neutral,  slightly 
alkaline,  or  more  infrequently  slightly  acid  reaction. 

Some  organisms  peptonize  milk  without  forming  a 
rennet  curd. 

(g)  Gas  Production.  This  is  characterized  by  the  for- 
mation of  gas  bubbles  in  the  milk,  and  is  generally  accom- 
panied by  the  formation  of  acid  curd.  Very  commonly  the 
curd  shrinks,  causing  extrusion  of  whey. 

EXERCISE  4.     PREPARATION  OF  LITMUS  MILK 

Apparatus.  Fresh  separated  or  skimmed  milk;  titra- 
tion  apparatus;  N/20  NaOH;  phenolphthalein  (indicator); 
5  c.c.  pipette;  azolitmin,  2%  solution;  filling  funnel; 
pinch  cock;  sterile  test  tubes;  apparatus  for  steam  sterili- 
zation. 

Method.  1.  Fresh  separated  or  skimmed  milk  should 
be  used.  Whole  milk  is  undesirable  on  account  of  its  fat 
content. 

2.  Titrate  and  record  the  reaction  of  the  milk.     If  the 
milk  titrates  above  17°  acid,  the  reaction  must  be  adjusted 
to  +15°.     Sour,  curdled  or  uncurdled  milk,  after  neutrali- 
zation,  does  not  make  a  desirable  nutrient  medium  for 
microorganisms,  therefore,  milk  whose  titre  is  above  20°-25° 
acid  should  be  discarded. 

Fresh  milk  varies  in  acidity  from  12°  to  18°.  Milk 
with  an  acidity  above  18°  to  phenolphthalein  will  not  give 
a  satisfactory  blue  color  with  azolitmin,  as  at  18°  it  begins 
to  show  the  acid  coloration. 

3.  Add  2%  of  a  standard  solution  of  Kahlbaum's  azo- 
litmin.    Litmus  or  azolitmin  is  added  merely  as  an  indi- 
cator and  should  be  of  sufficient  strength  so  as  not  to  dilute 
the  milk  to  any  extent. 


26  GENERAL  MICROBIOLOGY 

4.  Mix  the  milk  and  the  azolitmin  thoroughly  and  tube, 
using  approximately  8  c.c.  of  the  litmus  milk  in  each  tube. 

Note.  Care  should  be  taken  to  prevent  the  milk  from  coming  in 
contact  with  the  top  of  the  tubes,  as  it  will  cause  the  cotton  fibers  to 
adhere  to  the  tube.  This  may  be  avoided  by  the  use  of  a  "  filling 
funnel." 

5,  Sterilize   by   heating   in   flowing   steam  for  twenty 
minutes  on  four  successive  days.    Milk  is  difficult  to  sterilize, 
owing  to  the  resistant  spores  which  are  frequently  present. 
If  it  is  desired  to  sterilize  a  larger  bulk  than  in  tubes,  the 
time  of  heating  should  be  lengthened. 

Caution:  Overheating  tends  to  change  (caramelize)  the  milk 
sugar.  The  color  of  the  azolitmin  may  also  be  destroyed.  These 
changes  are  not  desirable. 

EXERCISE  5.    PREPARATION  OF  GLYCERIN  POTATO 

A  number  of  chromogenic  and  pathogenic  organisms 
thrive  especially  well  on  media  containing  glycerin.  The 
manner  in  which  glycerin  favors  the  growth  of  these  organ- 
isms is  not  known,  but  in  some  instances  it  seems  to  be 
directly  utilized  for  the  construction  of  fat  (Bact.  tubercu- 
losis) . 

Apparatus.  Large  healthy  potatoes;  cylindrical  potato 
knife,  or  cork  borer;  ordinary  knife;  tumbler;  sodium  car- 
bonate, 1  :  1000  solution;  glycerin,  5%  solution;  large 
sterile  test  tubes,  or  Roux  potato  tubes;  absorbent  cotton  or 
short  glass  rod;  1  c.c.  pipette;  distilled  water;  apparatus 
for  steam  sterilization. 

Method.     1.  Carefully  clean  one  or  two  large  potatoes. 

2.  By  means  of  a  cylindrical  potato  knife  or  cork  borer, 
cut  cylinders  of  potato,  4  to  6    cm.  long  and  1.5  to  1.8  cm. 
in  diameter.     With  an  ordinary  knife,  halve  each  cylinder 
by  a  diagonal  cut  so  that  each  piece  resembles  in  shape  an 
agar  slant.     Remove  any  portions  of  the  skin  on  these  pieces. 

3.  Place  in  a  tumbler  and  soak  in  a  dilute  (1  :  1000) 
solution  of  sodium  carbonate*  for  twenty-four  hours  only. 

*  Sodium  carbonate  is  used  to  neutralize  the  natural  acids  of  the  potato. 


PREPARATION  OF  GLYCERIN  POTATO 


27 


4.  Transfer  the  pieces  to  a  5%   solution  of  glycerin  in 
water  for  a  further  twenty-four  hours  only. 

5.  Place  in  sterile  tubes 
prepared  as  follows:  Select 
extra  large   test   tubes   1.5 
to   2  cm.   in  diameter  and 
clean  and  dry  them.     Place 
a  small  piece  of  absorbent 
cotton  or  glass  rod  0.5  cm. 
X2.5  cm.  in  the  bottom  of 
each.      Plug    with     cotton 
and  sterilize    in   the  usual 
way.      (Roux    tubes    need 
only    to    be    cleaned    and 
sterilized.) 

Just  before  introducing 
the  pieces  of  potato,  add 
about  1  c.c.  of  distilled  water 
to  each  tube,  using  a  pi- 
pette. The  potato  should 
not  touch  the  water. 

6.  Sterilize   by   heating 
at  100°  G.  on  four  successive 


days   for    twenty  minutes 
each  day. 


FIG.   8.— Potato  Tubes.      (Orig. 
Northrup.) 


Caution:  The  time  stated  in  3  and  4  must  be  strictly  adhered  to, 
else  the  potatoes  will  have  to  be  discarded  on  account  of  contamina- 
tion with  resistant  spore-forming  organisms. 


REFERENCE 

SMIRNOW,   M.  R.:    The  value  of  glycerinated  potato  as  a  culture 
medium.     Cent.  f.  Bakt.,  II  Abt.,  Bd.  41,  p.  303. 


28  GENERAL  MICROBIOLOGY 

EXERCISE   6.     PREPARATION   OF   MEAT   INFUSION 

Meat  infusion  is  the  foundation  of  the  ordinary  nutrient 
media,  as  broth,  gelatin  and  agar,  and  also  of  a  large  number 
of  special  nutrient  media,  as  sugar  broths,  etc. 

Under  these  directions  sufficient  meat  infusion  is  pre- 
pared to  make  1  liter  each  of  nutrient  broth,  gelatin  and 
agar. 

Apparatus.  1.5  kilograms  (3  Ibs.)  finely  chopped  fresh 
lean  beef;  1500  c.c.  tap  water;  3.5  liter  agateware  pail; 
large  funnel;  ring  stand;  clean  cloth;  1  liter  measuring 
cup;  three  sterile  1  liter  Erlenmeyer  flasks;  refrigerator; 
apparatus  for  steam  sterilization  (autoclav  preferable). 

Method.  1.  To  1.5  kilograms  of  finely  chopped,  fresh 
lean  beef  in  a  3.5  liter  agateware  pail,  add  1500  c.c.  of  tap 
water,*  mix  thoroughly  and  allow  to  stand  in  a  cool  place 
(refrigerator  preferred)  for  sixteen  to  twenty-four  hours  only. 

2.  Set  up  a  large  funnel  in  a  ring  stand  and  place  a  piece 
of  clean  cloth  in  the  funnel.     Place  a  measuring  cup  under 
the  funnel. 

3.  Strain  the  meat  infusion  through  clean  cheesecloth, 
thoroughly  pressing  out  all  the  juice.    1.5  liters  should  be 
recovered.     If  any  loss  occurs  make  up  to  1500  c.c.,  using 
tap  water. 

This  resulting  sanguineous  fluid  contains  the  soluble 
albumins  of  the  meat,  the  soluble  salts,  extractives  and  coloring 
matter,  chiefly  hemoglobin. 

4.  Place  500  c.c.  of  meat  infusion  in  each  of  three  sterile 
1  liter  Erlenmeyer  flasks.     Replace  the  plugs,  and  heat  in 
the  autoclav  at  120°  C.  for  thirty  minutes.     This  is  a  safer 
procedure  than  heating  for  a  longer  time  in  flowing  steam. 

During  this  heating  the  albumins  coagulable  by  heat  are 
precipitated. 

It  has  been  found  necessary  and  also  more  convenient 
to  prepare  and  sterilize  meat  infusion  before  proceeding  with 
the  preparation  of  the  different  media  in  which  it  is  used, 
*  Approximately  500  c.c.  of  water  to  each  pound  of  meat. 


PREPARATION  OF  NUTRIENT  BROTH  29 

on  account  of  the  resistant  spore-forming  organisms  which 
are  almost  universally  present  in  the  chopped  meat;  economy 
of  time  also  is  a  consideration.  Unless  sterilized  immedi- 
ately, meat  infusion  decomposes  quickly  owing  to  the 
abundance  and  diversity  of  the  microflora  acquired  during 
the  various  processes  of  preparation  for  market. 

Infusion  made  from  freshly  chopped  lean  beef  will  vary 
in  acidity  between  +15°  and  +25°  Fuller's  scale.  If  the 
reaction  is  markedly  lower  or  higher,  microbial  action  is 
taking  place,  which  is,  or  may  be,  injurious  to  the  food 
value  of  the  medium  in  which  the  meat  infusion  is 
used. 

The  infusion  contains  very  little  albuminous  matter  and 
consists  chiefly  of  the  soluble  salts  of  the  muscle,  certain 
extractives,  and  altered  coloring  matters  along  with  slight 
traces  of  protein  not  coagulated  by  heat. 

EXERCISE  7.  PREPARATION  OF  NUTRIENT  BROTH 

Nutrient  broth  is  the  standard  liquid  employed  for  cul- 
tivating microorganisms.  It  is  practically  a  beef  tea  con- 
taining peptone.  Peptone,  a  soluble  protein  not  coagulable 
by  heat,  is  added  to  replace  the  coagulated  albuminous 
substances  which  precipitate  when  the  meat  infusion  is 
sterilized.  Salt  is  added  to  take  the  place  of  the  phosphates 
and  carbonates,  some  of  which  are  precipitated  on  adjusting 
the  acidity  of  the  medium  by  sodium  hydroxide. 

The  reaction  of  ordinary  nutrient  media  is  adjusted  to 
about  +15°  with  phenolphthalein  as  indicator,  as  it  is  found 
that  most  microorganisms  grow  best  on  a  medium  neutral 
or  slightly  alkaline  to  litmus. 

When  it  is  required  that  nutrient  media  be  clear,  egg 
albumen  reduced  to  a  smooth  paste  with  water  (or  the  well- 
beaten  white  of  an  egg)  is  added.  By  coagulation,  the  egg 
albumen  removes  mechanically  all  small  particles  in  suspen- 
sion which  otherwise  would  pass  through  the  filter  paper. 


30  GENERAL  MICROBIOLOGY 

This  process  is  most  efficient  when  the  egg  albumen  coagu- 
lates slowly. 

As  egg  albumen  begins  to  coagulate  at  about  57°  C.  it  is 
absolutely  imperative  for  good  results  that  the  medium  be 
cooled  to  40°-50°  C.  before  the  addition  of  egg  albumen. 

Although  egg  albumen  contains  small  amounts  of  sol- 
uble matter  not  coagulable  by  heat,  as  sugar,  extractives 
and  mineral  matter,  all  of  which  will  serve  as  microbial 
food,  its  purpose  in  nutrient  media  is  primarily  for  its  clari- 
fying action. 

Apparatus.  500  c.c.  sterile  meat  infusion;  500  c.c.  tap 
water;  10  gms.  peptone,  Witte's;  5  gms.  salt;  10  gms.  egg 
albumen  (or  one  egg);  3.5  liter  agate-ware  pail;  titration 
apparatus;  N/20  NaOH;  N/l  NaOH;  phenolphthalein 
(indicator);  distilled  water;  5  c.c.  pipette;  large  stirring 
rod;  coarse  balances;  large  gas  burner;  large  funnel; 
plaited  filter  paper;  filling  funnel;  sterile  test  tubes;  sterile 
1  liter  flask;  apparatus  for  steam  sterilization-. 

Method.  1.  Put  the  contents  of  a  flask  of  meat  infusion 
(500  c.c.)  in  an  agate  pail  and  add  500  c.c.  of  tap  water. 

2.  Add  1%  of  Witte's  peptone  and  0.5%  of  salt. 

3.  Add  10  gms.  of  egg  albumen  which  has  been  well 
mixed  with  100  c.c.  of  tap  water.     (Put  the  egg  albumen 
in  a  tumbler  and  add  enough  water  to  form  a  paste.     Stir 
until  smooth.    Then  add  the  remaining  water.     One  egg* 
well  beaten  may  be  substituted.)     Mix  all  thoroughly. 

4.  Heat  in  flowing  steam  for  forty-five  minutes  or  in  the 
autoclav  at  120°  C.  for  thirty  minutes. 

5.  Titrate  with  N/20  NaOH. 

6.  Adjust  the  reaction  of  the  medium  to   +15°  with 
normal  NaOH  or  normal  HC1.     Retitrate  and  adjust  again 
if  necessary. 

7.  Counterpoise  and  note  the  weight. 

8.  Boil  fifteen  minutes  over  a  free  flame,  stirring  con- 
stantly. 

*  It  is  not  necessary  to  add  water  to  the  egg. 


GELATIN  31 

9.  Counterpoise  and  restore  any  loss  by  evaporation 
with  distilled  water. 

10.  Filter  while  boiling  hot  through  plaited  filter  paper 
just  previously  washed  with  1/2  liter  of  boiling  water. 

11.  Pass  the  filtrate  through  the  same  paper  till  it  is 
bright  and  clear. 

12.  Fill  thirty  sterile  test  tubes,   using  approximately 
8  c.c.  of  this  medium  for  each  tube.     Put   the  remaining 
broth  in  a  large,  sterile  flask. 

13.  Heat  the  test  tubes  and  contents  in  flowing  steam 
twenty  minutes  on  three  successive  days. 

14.  To  sterilize  a  large  flask  of  broth,  heat  for  twenty 
minutes  four  days  in  succession. 

GELATIN 

Gelatin  is  one  of  the  tools  of  the  microbiologist.  As 
such,  it  serves  two  purposes:  as  a  solid  culture  medium,  a 
technical  device  by  which  the  isolation  of  a  single  species 
of  microorganism  is  made  possible,  and,  to  those  organisms 
which  secrete  proteolytic  enzymes,  it  serves  as  a  nitrogenous 
food  material. 

Gelatin  bears  the  distinction  of  being  the  first  substance 
used  for  a  solid  culture  medium.  This  medium  was  origi- 
nated in  1882  by  Robert  Koch  and  has  since  revolutionized 
the  science  of  microbiology.  Prior  to  the  introduction  of 
solid  media,  the  isolation  of  a  single  species  of  microorganism 
involved  much  difficulty  and  almost  always  a  certain  measure 
of  uncertainty.  To  quote  from  Jordan:  "  It  cannot  be  a 
mere  coincidence  that  the  great  discoveries  in  bacteriology 
followed  fast  on  the  heels  of  this  important  technical 
improvement,  and  it  is  perhaps  not  too  much  to  claim  that 
the  rise  of  bacteriology  from  a  congeries  of  incomplete 
although  important  observations  into  the  position  of  a 
modern  biologic  science  should  be  dated  from  about  this 
period  (1882)." 

Koch's  first  plates  were  made  by  pouring  the  liquefied 


32  GENERAL  MICROBIOLOGY 

nutrient  gelatin  upon  sterile,  flat  pieces  of  glass.  The 
student  on  becoming  familiar  with  the  difficulties  of  pre- 
paring satisfactory  plates  with  the  use  of  the  "  Petri  dish  " 
will  appreciate  those  met  with  in  Koch's  first  gelatin  plates. 

Gelatin  is  a  protein,  i.e.,  a  nitrogenous  food  material. 
It  contains  as  its  essential  elements  carbon,  hydrogen, 
oxygen,  and  nitrogen  (other  elements,  however,  such  as 
sulphur,  phosphorus,  etc.,  may  be  present).  Its  empirical 
formula  according  to  Schiitzenberger  and  Bourgeois  is 
C7eHi24N24O29,  but  such  a  formula  only  gives  information 
of  the  chief  constituents  and  allows  one  to  form  some  idea 
of  the  huge  size  of  the  molecule;  no  idea  of  the  structure 
of  the  molecule  is  given.  However,  by  digesting  with 
dilute  sulphuric  acid,  gelatin  breaks  down  in  the  same  way 
as  the  proteins,  yielding  glycin,  leucin  and  other  fatty 
amino-acids. 

Gelatin  is  an  animal  protein,  but  does  hot  occur  as 
gelatin  in  the  animal  tissues.  It  exists  there  as  the  albu- 
minoid collagen  which  is  the  principal  solid  constituent  of 
fibrous  connective  tissue,  being  found  also,  but  in  smaller 
percentage,  in  cartilage,  bone  and  ligament.  Collagen  from 
these  various  sources  is  not  identical  in  composition  and 
gelatin  varies  correspondingly,  e.g.,  gelatin  from  cartilage 
differs  from  that  of  other  sources  in  that  it  contains  a  lower 
percentage  of  nitrogen. 

Gelatin,  the  body  resulting  from  the  hydrolysis  of 
collagen,  is  also  an  albuminoid.  (Hofmeister  regards  this 
hydrolysis  as  proceeding  according  to  the  equation  : 


collagen  +  water  =  gelatin 

but  in  dealing  with  substances  of  such  variable  composition, 

empirical  formulae  of  this  kind  have  no  great  significance). 

Commercially,  it  is  prepared  from  certain  kinds  of  bones 

and  parts  of  skin.     These  are  selected,  washed  and  extracted 


GELATIN  33 

by  water  and  with  a  dilute  acid  (hydrochloric),  with  rela- 
tively little  exposure  to  heat,  so  that  as  few  as  possible  of 
the  fluid  disintegration  products  of  the  stock  are  formed  and 
the  jellying  power  of  the  resultant  solution  is  not  destroyed. 

The  term  gelatin  is  derived  from  the  Latin  verb  gelare, 
to  congeal,  and  calls  to  mind  the  principal  attribute  of  this 
substance,  that  of  its  stiffening  or  jellying  property. 

Gelatin  belongs  to  that  interesting  class  of  substances 
called  colloids.  It  is  a  typical  example  of  the  class,  and 
exhibits  the  characteristic  properties  of  the  class.  Colloids, 
in  marked  contrast  to  crystalloids,  do  not  crystallize,  do 
not  readily  diffuse  and  are  impermeable  to  each  other. 
The  ultimate  particles  of  colloids  are  much  smaller  than 
what  we  would  ordinarily  term  a  physical  subdivision,  but 
rather  larger  than  chemical  molecules;  the  diameter  of  the 
smallest  particles  in  a  colloidal  solution,  e.g.,  red  colloidal 
gold,  which  have  been  counted  by  means  of  the  ultra-micro- 
scope, is  6  millimicrons  or  6  thousandths  of  a  micron.  A 
micron  is  one  thousandth  of  a  millimeter.  (Bacteria  are 
much  larger,  the  smallest  visible  by  means  of  the  ordinary 
microscope  being  from  0.3  to  1.0  micron  in  diameter.) 
Consequently  their  reactions  stand  midway  between  the 
physical  and  the  chemical  changes  of  matter,  as  may  be 
seen  by  considering  the  properties  of  gelatin. 

Gelatin  will  absorb  a  considerable  quantity  of  warm 
water  (it  is  almost  insoluble  in  cold  water)  and  swells  up, 
yielding  a  jelly  which,  upon  application  of  heat,  melts  to 
a  viscous,  sticky  solution  that  gelatinizes  again  upon  cooling. 
The  name  of  hydrogel  is  applied  to  colloids  showing  this 
property.  Ordinary  gelatin  media  for  microbiological  work 
contain  12%  to  15%  gelatin.  When  dried  at  medium  tem- 
peratures, gelatin  can  again  be  redissolved  and  redried  in- 
definitely. From  this  property  it  is  called  a  reversible 
colloid  to  distinguish  it  from  other  colloids  which,  when 
their  physical  state  is  once  changed,  are  insoluble,  e.g., 
casein  and  silicic  acid. 


34  GENERAL  MICROBIOLOGY 

If  superdried  at  about  130°  C.,  or  superheated  when  in 
the  gelatinous  state  either  for  a  short  time  at  a  temperature 
above  100°  C.,  or  for  a  long  time  at  100°  C.,  as  in  inter- 
mittent sterilization,  the  gelatin  is  so  modified  that  its 
redissolving  or  resolidifying  power  respectively  is  lost.  In 
superdrying,  the  loss  of  the  redissolving  property  is  laid  to 
the  too  close  contact  of  the  constituent  particles,  a  change 
in  the  physical  state;  in  the  superheated  gelatin,  the  loss  of 
the  resolidifying  power  is  probably  due  to  the  disintegra- 
tion of  the  gelatin  molecule,  a  more  purely  chemical 
phenomenon.  This  loss  of  the  gelatinizing  property  is  also 
caused  by  the  enzymic  activities  of  many  microorganisms 
and  is  also  a  disintegration  process. 

Gelatin  possesses  a  liquefaction  point  which,  however, 
varies  considerably  under  different  conditions.  Ordinarily, 
media  containing  12%  to  15%  gelatin  will  liquefy  or  melt 
at  a  temperature  in  the  vicinity  of  24°  to  26°  C.,  solidify- 
ing again  at  8°  to  10°  C.  to  a  clear,  transparent  jelly.  As  a 
consequence,  gelatin  media  may  be  employed  only  for 
organisms  which  do  not  require  a  higher  temperature  than 
22°  to  24°  C.  for  development.  Overheating  in  the  process 
of  preparation  or  sterilization  will  cause  a  considerable 
lowering  of  the  liquefaction  point,  perhaps  ultimately  so 
low  that  the  medium  will  be  liquid  at  room  temperature 
(20°  to  21°  C.)  It  will  readily  be  seen  how  the  latter 
gelatin  medium  could  not  handily  be  used  for  the  isola- 
tion of  organisms.  A  few  data  will  assist  in  fixing  this  in 
mind. 

The  solidifying  property  of  gelatin  varies  in  inverse 
proportion  with  the  time  of  heating  during  the  process  of 
sterilization;  its  liquefying  point  is  lowered  on  an  average  of 
2°  C.  for  each  hour  of  heating  at  100°  C.  This  makes  clear 
why  such  care  must  be  taken  in  the  preparation  of  a  gelatin 
medium,  in  the  fractional  sterilization  of  this  medium  in 
streaming  steam,  and  why  immediate  cooling  is  necessary 
aftei;  each  fractionation  in  the  process  of  its  preparation. 


GELATIN  35 

Although  temperatures  above  100°  C.  are  much  more 
destructive  to  the  solidifying  property  than  that  of  100°  C., 
it  is  possible  to  sterilize  a  medium  containing  12%  to  15% 
of  gelatin  in  the  autoclav  (7  to  8  Ibs.  pressure)  at  112°  to 
113°  C.  for  twenty  minutes  or  at  15  Ibs.  pressure  (120°  C. 
for  five  minutes)  without  impairing  its  usefulness  as  a  solid 
culture  medium. 

This  use  of  steam  under  pressure  (dry  steam)  is  almost 
necessary  in  the  case  of  a  gelatin  medium  to  effect  sterili- 
zation, since  gelatin,  from  its  source,  method  of  preparation, 
and  later  liabilities  to  contamination,  is  almost  certain  to 
contain  or  bear  upon  its  surface  a  large  number  of  very 
resistant  spores.  Heating  at  100°  C.  for  thirty  minutes 
on  three  or  even  four  or  five  consecutive  days  is  not  always 
efficient,  as  these  spores  do  not  always  germinate  within 
twenty-four  hours  after  heating  and,  referring  to  the  data 
above,  it  is  readily  seen  that  the  lowering  of  the  lique- 
faction point  is  not  to  be  considered  as  negligible  in  the 
process  of  intermittent  sterilization. 

Gelatin  possesses  another  property  which  renders  it 
valuable  for  bacteriological  work:  i.e.,  in  gelatin  plate 
cultures  no  water  of  condensation  ordinarily  collects  on  the 
cover  of  the  Petri  dish  (as  with  agar)  later  to  drop  on  the 
surface  of  the  gelatin  and  thus  obliterate  forms  of  colonies 
and  cause  isolated  colonies  to  become  contaminated  with 
neighboring  ones.  The  storing  of  this  medium  either 
in  test  tubes  or  in  plates,  sterile  or  inoculated,  is  thus 
rendered  much  more  simple  than  with  agar. 

REFERENCE 

VAN  DERHEIDE,  C.C.:  Gelatinose  Losungen  und  Verflussigungspunkt 
der  Nahrgelatine,  Arch.  f.  Hyg.,  Bd.  30,  1897,  pp.  82-115. 


36  GENERAL  MICROBIOLOGY 


EXERCISE  8.      PREPARATION  OF  NUTRIENT  GELATIN 

Apparatus.  500  c.c.  sterile  meat  infusion;  500  c.c.  tap 
water;  150  gms.  gelatin;  10  gms.  peptone,  Witte's;  5 
gms.  salt;  10  gms.  egg  albumen  (or  one  egg);  water  bath; 
thermometer;  3.5  liter  agate  ware  pail;  long  heavy  stirring- 
rod;  titration  apparatus;  N/20  NaOH;  N/l  NaOH; 
phenolphthalein  (indicator);  distilled  water;  coarse  bal- 
ances; large  gas  burner;  large  funnel;  plaited  filter  paper; 
filling  funnel;  sterile  test  tubes;  sterile  500  c.c.  Erlenmeyer 
flasks;  apparatus  for  steam  sterilization;  running-water 
bath  or  refrigerator. 

Method.  1.  Put  the  contents  of  a  flask  of  meat  infusion 
(500  c.c.)  in  an  agate  pail  and  add  500  c.c.  of  tap  water. 

2.  Add   15%  gelatin,   1%  Witte's  peptone,   and  0.5% 
salt  to  the  mixture. 

3.  Heat  this  mixture  in  a  water  bath  to  dissolve  the 
gelatin,  peptone  and  salt,  stirring  occasionally. 

4.  Cool  to  40°-50°  C.     This  is  imperative. 

5.  Then  add  10  gms.  of  egg  albumen  which  has  been  well 
mixed  with  100  c.c.  of  tap  water.     (Put  the  egg  albumen 
in  a  tumbler,  add  enough  water  to  form  a  paste  and  stir 
until  smooth;    then  add  the  remaining  water.     One  egg 
well  beaten  may  be  substituted.)     Mix  all  thoroughly. 

6.  Heat  in  flowing  steam  for  forty-five  minutes  or  in  the 
autoclav  at  105°  C.  for  thirty  minutes. 

7.  Titrate  with  N/20  NaOH. 

8.  Adjust   the  reaction  of  the  medium  to  +15°  with 
normal  NaOH  or  normal  HC1.     Re  titrate  and  adjust  again 
if  necessary. 

9.  Counterpoise  and  note  the  weight. 

10.  Boil  fifteen  minutes  over  the  free  flame,  stirring  con- 
stantly. 

11.  Counterpoise  and  restore  any  loss  by  evaporation 
with  distilled  water. 

12.  Filter  while  boiling  hot  through  plaited  filter  paper 


AGAR  37 

just  previously  washed  with  1/2  liter  boiling  water.  Pass 
the  filtrate  through  the  same  paper  until  it  is  bright  and 
clear. 

13.  Fill  thirty  sterile  test  tubes,  using  approximately 
8  c.c.  of  medium  for  each  tube.     Divide  the  remainder  into 
two  equal  portions  and  place  in  sterile  1/2  liter  Erlenmeyer 
flasks. 

14.  Heat   in  flowing   steam   twenty   minutes  on   three 
successive  days. 

15.  Cool  the  gelatin  in  a  running-water  bath,  immediately 
after  each  heating.     Care  must  be  taken  to  heat  the  gelatin 
as  little  as  possible,  since  part  of  the  solidifying  power  of 
gelatin  is  lost  with  each  application  of  heat. 

16.  To  sterilize  a  large  flask  of  nutrient  gelatin,  heat 
for  twenty  minutes  on  four  days  in  succession. 

AGAR 

Agar  or  agar-agar  (from  a  Malay  word  meaning  "  vege- 
table "),  the  substance  which  is  used  in  preparing  one  kind 
of  solid  culture  medium  for  bacteriological  work,  is  a  pro- 
duct prepared  from  various  seaweeds  found  near  the  Indian 
Ocean  and  in  Chinese  and  Japanese  waters.  This  type  of 
seaweed  has  several  common  names,  as  Ceylon  or  .Jaffna 
moss,  Bengal  isinglass,  etc.  Various  species  are  used  for 
food  and  the  trade  is  considerable. 

Payen,  a  French  chemist  -(about  1859),  obtained  the 
agar  jelly  from  the  seaweed,  Gelidium  corneum,  in  the  fol- 
lowing manner :  The  seaweed  was  allowed  to  stand  for  some 
time  in  a  cold  dilute  solution  of  hydrochloric  acid;  the  acid 
was  removed  by  rinsing  several  times  with  water,  then  the 
seaweed  was  placed  in  a  cold  dilute  solution  of  ammonia; 
next  the  ammonia  was  removed  by  repeated  rinsing  with 
cold  water.  During  this  process,  the  seaweed  lost  53% 
of  its  weight  in  mineral  salts,  coloring  matter,  and  organic 
constituents.  The  remaining  portion  was  boiled  in  water, 


38  GENERAL  MICROBIOLOGY 

during  which  process  the  vegetable  jelly  was  extracted. 
The  solution  so  obtained  was  poured  off,  leaving  the  useless 
sediment  behind.  This  jelly  is  the  same  in  composition 
as  that  existing  in  the  vegetable  tissues;  it  has  not  been 
changed  chemically,  as  is  collagen  in  the  preparation  of 
gelatin.  The  commercial  agar  is  most  probably  prepared 
by  evaporating  this  solution  to  dryness  by  different  means. 

Agar  usually  comes  into  the  hands  of  the  bacteriologist 
as  long,  slender,  grayish-white  strips,  or  as  blocks,  or  more 
especially  in  recent  years,  in  the  form  of  a  gray-white  pow- 
der of  European  manufacture. 

Agar,  in  contrast  with  gelatin,  is  a  carbohydrate,  i.e., 
it  consists  of  a  combination  of  carbon,  hydrogen  and  oxygen 
only.  Traces  of  nitrogen  are  present  as  impurities.  The 
above  qualitative  determinations  of  its  elementary  constit- 
uents were  made  by  Payen,  by  Parumbaru  and  by  Hueppe, 
who  made  their  determinations  on  agar  from  different 
sources.  As  far  as  can  be  ascertained,  its  empirical  formula 
has  not  yet  been  investigated  to  any  extent. 

Like  gelatin,  however,  agar  is  a  reversible  colloid.  It 
soaks  up  in  cold  water,  dissolves  in  hot  water  after  a  long 
boiling  to  a  tasteless  and  odorless  clear  solution,  and  solid- 
ifies upon  cooling  to  a  more  or  less  opaque  jelly.  Its 
watery  solution  is  neutral  or  nearly  neutral  to  phenol- 
phthalein;  still,  a  drop  or  two  of  twentieth  normal  sodium 
hydrate  is  sufficient  to  make  the  pink  color  perceptible. 

The  colloidal  properties  of  agar  are  not  destroyed  by  a 
long-continued  heating  at  a  high  temperature,  nor  by  the 
action  of  ordinary  microorganisms  as  are  those  of  gelatin. 
The  above  properties,  however,  are  influenced  and  may 
be  wholly  impaired  by  the  reaction  of  the  liquid  in  which 
the  agar  is  dissolved. 

The  reaction  of  the  liquid,  i.e.,  whether  it  is  acid  or 
alkaline,  influences  the  agar  as  to  its  solubility,  solidity, 
color,  transparency,  filterability  and  amount  of  condensa- 
tion water.  If  agar  is  dissolved  in  a  liquid  of  an  acidity 


AGAR  39 

equivalent  to  0.1%  HC1,  the  agar  dissolves  very  readily, 
filters  quickly,  the  resultant  filtrate  being  a  light  yellow, 
transparent,  slippery,  watery  solution  which  does  not 
solidify  upon  cooling.  If  a  smaller  percentage  of  hydro- 
chloric acid  is  used,  solidification  occurs  (below  40°  C.) 
but  the  jelly  will  not  "  stand  up  "  and  is  therefore  useless 
for  agar  slant  or  plate  cultures.  A  large  amount  of  con- 
densation water  is  present  also. 

If  agar  is  dissolved  in  a  weak  alkaline  or  neutral  broth, 
a  thick,  reddish-brown,  viscous  liquid  is  obtained  which 
filters  slowly  and  solidifies  quickly  at  40°  C.,  to  a  very  solid, 
opaque,  dry  jelly,  having  but  little  condensation  water; 
it  retains  its  shape  well  in  slants  and  in  plates.  Thus 
the  value  of  the  agar  as  a  solid  culture  medium  is  raised  or 
lowered  according  to  the  cjegree  of  alkalinity  or  acidity. 

It  must  be  noted  in  addition,  however,  that  when  once 
the  solidifying  property  of  agar  is  destroyed  by  the  presence 
of  an  excess  of  acid  in  its  solution,  this  property  can  never 
be  regained  by  neutralization  with  alkali;  the  acid  per- 
manently destroys  the  reversibility  of  the  colloid. 

The  melting-point  of  agar  (of  1.5%  in  neutral  solution) 
is  97°  C.  and  although  its  solidifying  point  is  at  40°  C., 
when  once  it  has  solidified  it  will  stand  up  in  the  thermostat 
at  a  temperature  of  50°  C.  For  bacteriological  purposes, 
only  that  form  of  agar  can  be  used  which  remains  fluid  at 
from  38°  to  40°  C.  Agar  which  remains  fluid  only  at  a 
temperature  above  this  point  would  be  too  hot  when  in 
a  fluid  state  for  use;  the  vitality  of  organisms  introduced 
would  be  impaired  or  destroyed  by  the  high  temperature. 

Difficulties  are  encountered  in  the  preparation  of  a  solid 
culture  medium  from  agar,  due  to  its  slow  solubility,  vis- 
cosity and  consequent  slow  filterability.  Its  solution 
(digestion)  is  effected,  as  mentioned  above,  by  a  long 
heating  in  a  water-bath,  steam  sterilizer,  autoclav,  or  over 
a  free  flame.  The  length  of  time  required  for  complete 
digestion  depends  upon  three  things:  The  reaction  of  the 


40  GENERAL  MICROBIOLOGY 

liquid  in  which  the  agar  is  dissolved,  the  per  cent  content 
of  agar,  and  the  method  of  dissolving.  The  influence  of  the 
reaction  of  agar  solutions  has  been  treated  above.  For 
general  culture  use,  however,  ordinary  agar  is  made  +15° 
Fuller's  scale  (agar  solidifies  with  difficulty  above  +30° 
Fuller's  scale). 

One  per  cent  agar  is  much  more  easily  soluble  under 
equal  conditions  than  a  higher  per  cent.  One  and  one- 
half  per  cent  is  the  amount  used  in  ordinary  agar  media, 
giving  a  somewhat  stiffer  and  thus  more  desirable  jelly. 

Agar  is  digested  most  rapidly  over  a  free  flame.  If  not 
heated  sufficiently,  after  the  filtration  and  sterilization  of 
the  agar  by  the  intermittent  method,  a  flocculent  precip- 
itate frequently  appears  in  the  previously  clear  medium. 
This  can  be  made  to  disappear  in  most  cases  by  subjecting 
to  the  temperature  of  the  autoclav  (120°  C. — 15  Ibs.). 

Agar  for  culture  media  should  be  entirely  clear  when 
liquid,  and  homogeneously  opaque-translucent  when  solid; 
it  should  have  a  translucence  sufficient  to  allow  deep  colonies 
on  plates  or  stab  cultures  to  be  observed  readily;  it  should 
not  contain  flocculent  material,  sediment,  or  pieces  of  cotton 
or  filter  paper,  as  these  hinder  typical  colony  development 
of  microorganisms  and,  to  the  inexperienced,  may  some- 
times be  mistaken  for  colonies. 

In  the  first  methods  ever  used  for  making  agar  culture 
media,  instead  of  filtering  the  hot  agar  through  filter  paper, 
absorbent  cotton,  or  asbestos,  it  was  allowed  to  cool,  dur- 
ing which  process  the  sediment  settled  to  the  bottom;  when 
solid  the  sediment  was  cut  off.  This  method  was  not 
desirable,  as  the  clearness  of  the  resultant  agar  would  depend 
upon  the  rate  of  cooling;  the  slower  the  cooling,  the  more 
completely  would  sedimentation  take  place. 

Agar  is  not  a  food  for  microorganisms  in  general,  i.e., 
it  is  not  affected  by  the  digestive  enzymes  of  most  bacteria, 
as  is  gelatin.  However,  a  few  bacteria  are  known  which 
have  the  power  of  liquefying  agar,  among  which  are  B. 


PREPARATION  OF  NUTRIENT  AGAR  41 

gelaticus  n.  sp.  (gran)  and  Bad.  nenckii,  both  of  which  are 
found,  as  would  be  expected,  in  sea  water.  This  compara- 
tive inertness  of  agar  renders  it  valuable  for  the  preparation, 
of  solid  synthetic  media,  the  value  of  which  may  be  en- 
hanced by  subjecting  the  commercial  agar  to  natural 
fermentation  during  which  process  any  traces  of  avail- 
able food  substances  are  used  up  by  the  microorganisms 
present.  (Beijerinck.) 

Agar  is  of  special  use  in  bacteriological  work  in  which 
the  cultivation  of  microorganisms  must  be  conducted  at  a 
temperature  above  the  melting-point  of  gelatin.  This 
feature  has  made  possible  the  great  strides  that  have  been 
taken  in  medical  bacteriology,  as  many  pathogenic  bacteria 
can  be  isolated  and  grown  only  with  difficulty  at  tempera- 
tures below  that  of  the  body. 

REFERENCES 

SMITH,  ERWIN  F.:    Bacteria  in  Relation  to  Plant  Diseases.     Vol.  I, 

pp.  31-36.     Several  illustrations. 
SCHULTZ,  N.  K.:  Zur  Frage  von  der  Bereitung  einiger  Nahrsubstrate. 

Cent.  f.  Bakt.  I.  Orig.,  Bd.  10,  1891,  p.  57. 

EXERCISE  9.     PREPARATION  OF  NUTRIENT  AGAR 

Apparatus.  3.5  liter  agate-ware  pail;  15  gms.  agar; 
10  gms.  peptone;  5  gms.  salt;  10  gms.  egg  albumen  (or  one 
egg);  500  c.c.  sterile  meat  infusion;  500  c.c.  tap  water; 
titration  apparatus;  N/20  NaOH;  N/l  NaOH;  phenol- 
phthalein  (indicator);  distilled  water;  large  funnel; 
plaited  filter  paper;  filling  funnel;  sterile  test  tubes; 
sterile  liter  flask;  coarse  balances;  large  gas  burner;  1 
liter  measuring  cup;  apparatus  for  steam  sterilization. 

Method.  1.  In  a  3  liter  agate  ware  pail  place  15  gms. 
of  agar  in  500  c.c.  of  tap  water. 

2.  Wash  the  agar  well,  separating  the  shreds  and  squeez- 
ing it  through  the  hands. 

3.  Decant  the  dirty  water,  measuring  the  amount  poured 


42 


GENERAL  MICROBIOLOGY 


off;    replace  with  the  same  amount  of  clean  tap  water. 

Repeat. 

4.  Dissolve  over  a  free  flame  and  boil  for  five  minutes, 
stirring  constantly.  The  solu- 
tion must  be  entirely  free  from 
lumps  of  agar. 

5.  Add  1%  Witte's   peptone 
and    0.5%   salt   to  the   boiling 
agar. 

6.  To  500  c.c.   of    meat  in- 
fusion add   10  gms.    of  egg  al- 
bumen   which    has    been    well 
mixed  with  100  c.c.  of  tap  water. 
(Put    the    egg    albumen    in    a 
tumbler  and  add  enough  water 
to    form    a    paste.      Stir    until 
smooth  and  then  add  the  remain- 
ing water.    One  egg;  well  beaten, 

FIG.  9.— Hot  Water  Funnel  for   may   be    substituted.)       Mix   all 
Filtering  Agar  or  Gelatin.       thoroughly 

7.  Pour  the  melted  agar  mixture  slowly  into  the  meat 
infusion,    stirring    constantly.     Heat    in    the  autoclav  at 
120°  C.  for  forty-five  minutes  or  for  an  hour  in  flowing 
steam. 

Note.  The  time  for  this  heating  may  be  lengthened  to  advantage, 
but  never  shortened.  If  agar  has  not  been  heated  sufficiently  before 
filtration,  a  flocculent  precipitate  will  form  in  the  tubes  upon  heating 
in  flowing  steam.  In  most  cases  this  may  be  caused  to  disappear  by 
heating  for  a  short  time  in  the  autoclav  at  15  Ibs. 

8.  Titrate  with  N/20  NaOH. 

9.  Adjust  the  reaction   of  the  medium  to  +15°  with 
normal   NaOH   or   normal   HC1.     Retitrate   and   readjust 
the  reaction  if  necessary. 

10.  Counterpoise  and  note  the  weight. 

11.  Boil  fifteen  minutes  over  a  free  flame,  stirring  con- 
stantly, 


DUNHAM'S  PEPTONE   SOLUTION  43 

12.  Counterpoise  and  make  up  any  loss  in  weight  with 
boiling  distilled  water. 

13.  Filter  boiling  hot  through  plaited  filter  paper  just 
previously   washed   with   boiling   water.     Pass   the   filtrate 
through  the  same  paper  until  clear. 

14.  Fill  60  to  70  sterile  test  tubes,  using  approximately 
8  c.c.  of  the  medium  for  each  tube. 

15.  Heat  in  flowing  steam  twenty  minutes  on  three  suc- 
cessive days. 

16.  At  the  end  of  the  final  heating,  place  the  tubes  of 
agar  in  an  inclined  position  to  solidify  (do  not  allow  the 
medium  to  touch  the  plug)  so  that  a  large  surface  is  pre- 
sented for  the  cultivation  of  microorganisms.     These  are 
called  agar  slants. 

Note.     If  agar  tubes  are  to  be  used  only  for  agar  slants,  less  of  the 
medium  is  needed  in  the  tube  than  when  they  are  to  be  used  for  plating. 

17.  To  sterilize  a  large  flask  of  agar,  heat  for  thirty 
minutes  on  four  successive  days. 

EXERCISE  10.     PREPARATION  OF  DUNHAM'S  PEPTONE 
SOLUTION 

Dunham's  solution  is  utilized  for  determining  the 
power  of  microorganisms  to  produce  indol,  ammonia  or 
nitrites  from  peptone,  which  properties  are  character- 
istic of  certain  species. 

Apparatus.  1000  c.c.  of  tap  water;  10  gms.  peptone, 
Witte's;  5  gms.  salt;  large  burner;  large  funnel;  plaited 
filter  paper;  filling  funnel;  sterile  test  tubes;  apparatus 
for  steam  sterilization. 

Method.  1.  Mix  1%  peptone  and  0.5%  salt  to  a 
smooth  paste  with  a  measured  (small)  amount  of  water. 

2.  Dilute  to  1000  c.c.  with  tap  water. 

3.  Counterpoise  and  note  the  weight. 

4.  Boil  ten  minutes  over  a  free  flame;   counterpoise  and 
make  up  any  loss  in  weight  with  distilled  water. 


44  GENERAL  MICROBIOLOGY 

5.  Filter  while  hot  through  a  plaited  filter  previously 
washed  with  hot  water.     (Filtrate  must  be  perfectly  trans- 
parent.) 

6.  Tube,  putting  8  c.c.  in  each  tube. 

7.  Sterilize    for    fifteen    minutes    on    three    successive 
days. 

Microorganisms  which  will  not  produce  ammonia  or 
nitrites  from  peptone  may  show  this  power  if  nitrogen  is 
added  to  this  solution  in  the  form  of  inorganic  nitrogen  as 
potassium  nitrate  (0.2%). 

EXERCISE  11.     NITRATE  PEPTONE  SOLUTION 

This  solution  is  used  to  determine  the  power  some 
organisms  have  of  reducing  nitrates  to  nitrites,  free  ammonia 
or  nitrogen. 

Apparatus.  1000  c.c.  distilled  water;  1  gm.  peptone, 
Witte's;  0.2  gm.  nitrite-free  potassium  nitrate;  'large  agate- 
ware pail;  filling  funnel;  sterile  test  tubes;  apparatus  for 
steam  sterilization. 

Method.  1.  Mix  the  following  ingredients:  1000  c.c. 
distilled  water;  1  gm.  Witte's,  or  other  peptone;  0.2  gm. 
nitrite-free  potassium  nitrate.  Filter  if  necessary, 

2.  Tube,  placing  8  c.c.  in  each  tube. 

3.  Sterilize  by  heating  for  fifteen  minutes  on  three  suc- 
cessive days  or  for  five  minutes  in  the  autoclav  at  120°  C. 

CULTURES 

Definitions.  A  culture  consists  of  the  active  growth 
of  microorganisms  in  or  on  a  nutrient  medium. 

A  mixed  culture  is  a  culture  composed  of  two  or  more 
species  of  microorganisms  growing  together  in  or  on  a 
nutrient  medium. 

A  pure  culture  is  the  growth  of  one  species  of  micro- 
organism only,  in  or  on  a  nutrient  medium,  that  was  sterile 
before  inoculation. 


CULTUKES  45 

Pure  cultures  are  used  for  studying  the  morphological 
and  physiological  characteristics  of  microorganisms. 

From  mixed  cultures,  pure  cultures  may  be  obtained  by 
the  plating  method.  Mixed  cultures  of  known  micro- 
organisms may  be  employed  in  studies  on  symbiosis,  meta- 
biosis,  or  antibiosis. 


FIG.    10.— Mixed   Culture   in   Petri   Dish    (Plate   Culture)    Showing 
Various  Forms  and  Sizes  of  Colonies,     (Orig,  Northrup.) 

Plate  cultures  are  cultures  grown  in  Petri  dishes  contain- 
ing a  nutrient  medium. 

Slant  culture  is  the  term  generally  applied  to  cultures 
grown  on  the  inclined  surface  of  any  medium,  such  as  agar, 
potato,  blood  serum,  etc.,  and  are  designated  specifically 
as  agar  slant  cultures,  potato  slant  cultures,  etc.  They  are 
generally  prepared  by  drawing  a  contaminated  needle  in 
a  straight  line  along  the  surface  of  the  medium.  Cul- 


46 


GENERAL  MICROBIOLOGY 


tures  prepared  in  this  way  are  also  frequently  termed 
streak  cultures.  The  term  streak  cultures  may  also  be 
applied  to  cultures  made  similarly  but  grown  on  a  horizon- 
tal flat  surface  as  in  a  Petri  dish. 

Slant  or  streak  cultures  are  valuable  in  offering  a  large 
surface  for  growth,  to  aerobic  organisms. 

Stab  (or  Stich)  culture  is  the 
term  applied  to  a  culture,  generally 
a  pure  culture,  which  is  prepared 
by  stabbing  a  translucent,  liquefi- 
able  solid  medium  to  a  considerable 
depth  with  a  contaminated  straight 
needle.  Gelatin  stab  cultures  are 
invaluable  for  studying  gelatin 
liquefaction.  Agar  is  frequently 
used  for  stab  cultures.  If  sugar  is 
added  to  the  medium,  gas  produc- 
tion may  be  demonstrated.  Aerobic 
and  anaerobic  bacteria  may  be 
easily  differentiated  by  their  be- 
havior in  stab  culture. 

Liquid  cultures  are  cultures 
grown  in  a  liquid  medium  such  as 
milk,  broth,  cider,  wort,  etc. 

Shake    cultures   are   made   by 

FIG.  11.— Liquefaction  of  inoculating  with  a  pure  or  mixed 
culture,  a  liquefied  nutrient  me- 
dium (40°-45°  C.).  The  inoculum 
is  distributed  immediately  through- 
out the  medium  by  means  of  the  needle  used,  or  by  rotat- 
ing or  shaking. 

This  type  of  cultivation  is  valuable  for  determining  the 
oxygen  relation  of  the  organisms  introduced  and  is  espe- 
cially useful  for  demonstrating  the  presence  of  gas-producing 
organisms  if  a  suitable  medium  is  used. 

Care  of  Cultures.    1.  Incubation:    Cultures  should  b.§ 


Gelatin,  Saccate  becom- 
i  n  g  Infundibuliform. 
(Orig.  Northrup.) 


CULTURES 


47 


kept  at  a  constant  temperature.     Organisms  which  natur- 
ally grow  at  body  temperature  (37°  C.)  as  Bacillus  coli, 


I 


a 
I 


1 


Streptococcus   pyogenes,    etc.,    may,    w#Ji   rffte   exception   of 
gelatin  cultures,  be  kept  in  the  37°  C.  incubator. 

Always  place  cultures  in  tumblers  with  cotton  in  the 
bottom  or  in  small  wire  baskets;    never  place  them  in  a 


48  GENERAL  MICROBIOLOGY 

horizontal   position   or   incline   them   carelessly   against   a 
vertical  surface  without  proper  support. 

2.  Care  of  Broken  Cultures.    If  a  culture  of  any  organism 
is  accidently  broken  pour  1  :  1000  mercuric  chloride,  2% 
compound  solution  of  cresol  or  5%  phenol  over  it  and  also 
over  any  articles  which  may  have  been  infected;    let  stand 
ten  minutes  before  wiping  up.     Always  disinfect  your  hands 
after  handling  broken  cultures. 

3.  Disposal  of  Old  Cultures.     Heat  glassware  contain- 
ing cultures  to  be  discarded  one  hour  in  flowing  steam. 
Cultures  of  pathogenic  spore-forming  organisms  should  be 
autoclaved.     Glassware  so  treated  may  safely  be  washed  by 
the  student. 

Never  throw  living  cultures  into  waste  crocks,  sinks, 
or  elsewhere.  You  safeguard  yourself  and  others  in  the 
laboratory  by  destroying  all  living  cultures.  Carelessness 
in  regard  to  this  matter  will  not  be  tolerated. 

4.  Care  of  Slides,  Cover-glasses,  etc.      Slides  and  cover- 
glasses   used   for   hanging   drop   mounts,    etc.,    should   be 
immersed  in   1  :  1000  mercuric   chloride  or  chromic   acid 
cleaning  solution  for  at  least  ten  minutes  before  cleaning. 

5.  Care  of  Cuts  and  Other  Wounds.     In  case  of  cuts  or 
wounds,  consult  the  instructor  at  once.     All  wounds  should 
be  attended  to  immediately.     Tincture  of  iodin  is  recom- 
mended for  painting  skin  abrasions  and  deep  wounds;   in 
the  latter  case  a  bandage  should  be  applied  to  keep  extra- 
neous matter  from  entering  and  setting  up  infection.     In 
case  of  serious  injury,   a  physician  should  be  consulted. 
Every  laboratory  should  keep  a  stock  of  rolled  bandages, 
etc.,  for  emergencies, 


PREPARATION  OF  PLATE  CULTURES 


49 


EXERCISE  12.  PREPARATION  OF  PLATE  CULTURES, 
LOOP  OR  STRAIGHT-NEEDLE  DILUTION  METHOD 
(QUALITATIVE) 

Plate  cultures  are  a  valuable  asset  to  the  microbiologist, 
as  they  offer  a  means  by  which  pure  cultures  of  micro- 
organisms may  most  easily  be  obtained;  they  also  allow 
a  quantitative  and  qualitative 
study  of  the  micron1  ora  of  differ- 
ent substances. 

Their  preparation  consists  in, 
(1)  inoculating  a  liquefied  solid 
culture  medium  with  micro- 
organisms, (2)  mixing  them  well 
throughout  the  medium,  (3) 
pouring  the  inoculated  medium 
into  a  sterile  Petri  dish  and, 
when  it  has  solidified,  (4)  placing 
the  Petri  dish  or  plate  culture 
at  a  constant  temperature. 

The  culture  medium  in  so- 
lidifying fixes  in  situ  the  micro- 
organisms introduced,  and  well- 
separated  organisms  develop  into 
more  or  less  well-separated 
"  colonies  "  which  become  visible 
to  the  naked  eye  after  twenty- 
four  to  forty-eight  hours.  From  FlG- 13. -Water-bath  for  Melt- 
these  isolated  colonies  usually 
pure  cultures  may  then  be  ob- 
tained, or  a  quantitative  or  quali- 
tative study  may  be  made. 

Isolated  surface  colonies  are  most  frequently  round 
(concentric  in  growth)  and  generally  are  quite  typical  for 
each  species,  while  isolated  sub-surface  colonies  are  lenticu- 


ing  Agar  or  Gelatin  for  Plat- 
ing, containing  a  Removable 
Copper  Test  Tube  Rack. 
(Orig.  Northrup.) 


50 


GENERAL  MICROBIOLOGY 


lar  (double  concave)  or  compoundly  lenticular  in  shape  as  a 
rule,  species  differences  not  being  as  well  denned. 

Apparatus.     Tripod  leveling  stand;    glass  plate  about 


I 


14  inches  square;  small  spirit  level;  water-bath;  thermom- 
eter; sterile  Petri  dishes;  tubes  of  sterile  media  (gelatin 
or  agar);  culture;  platinum  needle  and  loop;  Bunsen 
burner;  wax  pencil;  mixed  or  pure  culture. 


PREPARATION  OF  PLATE  CULTURES  51 

I.  Procedure  for  Agar  Plates.  The  loop  or  straight- 
needle  dilution  method  is  valuable  as  a  quick  method  of 
obtaining  pure  cultures  when  quantitative  results  are  not 
desired. 

1.  Place  the  glass  plate  on  the  leveling  stand. 

2.  Place  the  spirit  level  on  the  glass  plate  and  make 
level  by  means  of  the  leveling  screws. 

Note.  The  plate-leveling  stand  facilitates  the  uniform  distribution 
of  the  medium  over  the  bottom  of  the  Petri  dish,  but  is  not  necessary 
for  the  accomplishment  of  favorable  results.  If  the  desk  top  is  level 
this  apparatus  is  unnecessary. 

3.  Place  three  sterile  Petri  dishes,  labeled  1,  2  and  3, 
in  a  row  on  the  glass  plate. 

4.  Liquefy  three  tubes  of  agar  at  100°  C.  in  the  water- 
bath  or   steam    and    keep  at   a  temperature    of    40°   to 
45°  C. 

5.  Number  the  tubes  of  agar  1,  2  and  3  and  flame  the 
plugs. 

6.  With   the   sterilized   platinum  needle,   merely  touch 
the  culture  and  transfer  to  tube  No.  1. 

Note.  Hold  cultures  and  plugs  while  transferring  as  in  Fig.  20, 
p.  59. 

7.  Distribute  the  microorganisms  through  the  medium 
with  the  needle. 

8.  Transfer  one  loopful  from  tube  No.  1  to  tube  No.  2 
and  mix  with  the  needle,  as  in  7. 

9.  Slightly  raise  the  cover  of  Petri  dish  No.  1.     Intro- 
duce the  flamed  mouth  of  tube  No.  1  and  pour  the  melted 
agar  into  the  plate;    remove  the  mouth  of  the  tube,  and 
replace  the  cover  of  the  Petri  dish.     If  the  medium  has 
not  entirely  covered  the  bottom  of  the  plate,  tilt  slightly 
in  different  directions  to  distribute  evenly. 

Note.  Passing  the  Petri  dish  several  times  through  the  flame  just 
previous  to  pouring  the  plate  will  aid  greatly  in  preventing  the  forma- 
tion of  condensation  water  on  the  cover. 


52  GENERAL  MICROBIOLOGY 

10.  Transfer  two  loopfuls  from  tube  No.  2  to  tube  No. 
3  and  mix. 

11.  Plate  tube  No.  2  in  Petri  dish  No.  2  (see  9). 

12.  Plate  tube  No.  3. 

13.  Label  the  plates  with  name  of  culture,  number  of 
dilution  and  date,  and  with  your  own  name  or  desk  number. 

14.  When   the    agar   has    solidified    firmly,    invert   the 
plates  and  place  in  the  incubator  at  37°  C.,  or  at  room 
temperature. 

Note.  If  the  plates  are  placed  right  side  up,  condensation  water 
forms  on  the  cover  and  drops  down  upon  the  surface  of  the  agar,  caus- 
ing the  colonies  to  run  together  and  thus  destroying  their  character- 
istic appearance. 

II.  Procedure  for  Gelatin  Plates. 

1-3.  Proceed  as  in  I.  "  Procedure  for  Agar  Plates." 

4.  Liquefy   three   tubes   of  gelatin   in   the   water-bath 

and  keep  at  a  constant  temperature  of  30°  to  35°  C. 
5-13.  Proceed  as  in  I.  "  Procedure  for  Agar  Plates." 
14.  Place  at  a  constant  temperature  of  21°  C.     The 

gelatin  may  not  harden  until  placed  at  this  temperature. 

Note.  Gelatin  plates  are  kept  right  side  up,  as  the  organisms  may 
liquefy  the  gelatin.  The  liquefied  part  would  then  fall  from  the  medium 
upon  the  cover  and  ruin  the  plate  for  study. 

EXERCISE   13.     PREPARATION    OF   PLATE    CULTURES, 
QUANTITATIVE  DILUTION  METHOD 

In  the  method  given  below,  "  dilution  flasks  "  are  pre- 
pared containing  measured  amounts  of  water  or  salt  solu- 
tion in  which  a  measured  amount  of  the  substance  under 
investigation  is  placed. 

As  to  whether  water  or  salt  solution  is  used  depends  upon 
the  nature  of  the  material  to  be  dissolved  or  placed  in 
suspension.  If  the  substance  whose  microflora  is  to  be 
studied  contains  a  certain  amount  of  various  salts  or  other 
electrolytes  in  solution,  an  effort  should  be  made  to  approx-* 


QUANTITATIVE  DILUTION  METHOD 


53 


imate  this  amount  in  the  preparation  of  the  diluting  fluid, 
e.g.,  in  obtaining  a  quantitative  estimation  of  the  micro- 


FIG.  15. — Incubator. 


organisms  from  the  blood,  dilutions  should  be  made  in  0.85% 
salt  solution;  from  tap  water,  in  tap  water,  etc. 

Theoretically,  dilutions  made  in  a  liquid  of  a  markedly 
different  electrolyte  concentration  from  that  of  the  sub 


54 


GENERAL  MICROBIOLOGY 


stance  to  be  studied,  might  cause  either  plasmolysis  or 
plasmoptysis  as  the  concentration  was  respectively  too 
great  or  too  weak. 

Microorganisms  of  different  species  differ  markedly 
in  their  susceptibility  to  osmotic  pressure.  This  cannot 
be  determined,  however,  unless  studies  are  made  of  pure 
cultures  of  each,  therefore  the  percentage  of  salt  in  the 
diluting  liquid  should  approximate 
that  of  the  substance  whose  micro- 
flora  is  to  be  studied. 

The  method  below  is  applicable 
to  substances  in  the  liquid  condi- 
tion only.  Modifications  of  this 
method  may  be  utilized  to  apply  to 
nearly  every  class  of  substances. 

Plates  are  generally  made  from 
three  different  dilutions,  so  that 
well-separated  colonies  may  be  ob- 
tained on  at  least  two  plates. 

Apparatus.  Sterile  1  c.c.  pipettes 
(graduated  to  0.1  c.c.);  sterile 
10  c.c.  pipettes  (graduated) ;  sterile 

FIG.  "16. -Koch's   Safety  Erlenmeyer  flasks  of   200  c.c.   ca- 
pacity containing  90  c.c.  and  99  c.c. 
of  sterile   water   or  salt    solution; 
three  sterile  Petri  dishes;  three  tubes  of  sterile  agar  or  gelatin. 


Burner  for  Incubator  or 
Water  Bath. 


Note.  Use  only  freshly  prepared  dilution  flasks,  otherwise  evapora- 
tion takes  place  so  rapidly  that  accuracy  is  not  possible. 

Culture.     Substance  under  investigation. 

Method.  1.  With  a  sterile  1  c.c.  pipette,  transfer  1  c.c. 
of  the  original  sample  or  culture  to  a  flask  containing  99 
c.c.  of  sterile  water  or  salt  solution.  The  flask  now  con- 
tains 100  c.c.  of  liquid  containing  1  c.c.  of  the  original 
sample,  giving  a  dilution  of  1  in  100. 

Note.    A  sterile  pipette  must  be  used  for  each  separate  operation. 


QUANTITATIVE  DILUTION  METHOD  55 

2.  Shake  the  flask  to  secure  an  even  suspension  of  the 
microorganisms. 

Do  not  allow  the  liquid  to  touch  the  cotton  plug. 

3.  With  a  sterile  pipette,   transfer   1   c.c.   of  the  first 
dilution  into  a  flask  containing  99   c.c.   of  sterile  water 
and  shake.     The  second  flask  now  contains  100  c.c.  of  a 
liquid  containing  1/100  of  the  original  sample,  a  dilution 
of  1/100  in  100,  or  1  in  10,000. 

4.  If  a  higher  dilution  is  required,  1  c.c.  from  the  flask 
containing  the   1/10,000   dilution   placed   in   a  flask  con- 
taining 99  c.c.  sterile  water  gives  100  c.c.  of  a  liquid  con- 
taining 1/10,000  of  the  original  sample,  or  a  dilution  of 
1  in  1,000,000. 

If  a  lower  dilution  of  the  original  sample  than  1/100 
is  desired,  make  use  of  the  90  c.c.  dilution  flasks  as  follows: 

With  a  sterile  10  c.c.  pipette  place  10  c.c.  of  the  original 
sample  into  90  c.c.  of  sterile  water  and  shake.  This  flask 
now  contains  100  c.c.  of  liquid  containing  10  c.c.  of  the 
original  sample,  giving  a  dilution  of  1  in  10.  A  dilution 
of  1  in  1000  may  be  made  either  by  placing  1  c.c.  of  the  1/10 
dilution  in  99  c.c.  of  sterile  water,  or  by  placing  10  c.c.  of 
the  1/100  dilution  in  90  c.c.  of  sterile  water. 

Note.  Almost  any  desired  dilution  can  be  made  by  the  use  of  these 
flasks. 

5.  For  plating,  transfer  1  c.c.  with  a  sterile  1  c.c.  pipette 
from  the  flask  containing  the  desired   dilution  to  a  sterile 
Petri  dish. 

Note.  Never  use  less  than  1  c.c.  Run  duplicates  when  absolute 
accuracy  is  necessary. 

6.  Liquefy  the  desired  number  of  agar  or  gelatin  tubes 
in  the  water-bath  or  steam  at  100°  C. 

7.  Cool  to  a  temperature  of  40°  to  45°  C. 

8.  Pour  the  plates,  tilting  each  carefully  so  that  the 
1  c.c.  of  the  diluted  sample  may  be  mixed  well  throughout 
Jhe  medium. 


56 


GENERAL  MICROBIOLOGY 


9.  Place  the  plates  on  a  level  surface  until  the  medium 
solidifies. 

10.  Incubate  at  the  desired  temperature. 

EXERCISE  14.     METHODS    OF    COUNTING   COLONIES 
IN  PETRI  DISH  CULTURES 

Apparatus.  Jeffer's  counting  plate;  black  glass  plate  or 
cardboard;  tripod  counting  lens,  magnifying  four  diam- 
eters; plate  cultures. 

Note.  In  Jeffer's  counting  plate  (see  illustration)  each  division 
has  an  area  of  1  square  centimeter.  The  figures  denote  the  number 
of  square  centimeters  in  the  respective  circles. 


123156789  10 


FIG.  17. — Jeffer's  Counting 
Plate. 


FIG.  18.— Wolfhugel'g 
Counting  Plate. 


Method.  1.  Invert  the  Petri  dish  culture  to  be  counted 
upon  the  black  glass  plate  or  upon  some  black  surface. 

Note.  If  liquefiers  are  present  on  the  gelatin  plate,  place  the  Petri 
dish  right  side  up  upon  the  counting  plate;  this  necessitates  refocusing 
the  lens.  The  cover  may  be  removed  to  facilitate  counting  if  the 
plate  is  to  be  discarded. 

2.  Place  the  counting  plate  upon  the  Petri  dish,  making 
the  circumference  of  the  Petri  dish  coincide  as  nearly  as 


COUNTING  COLONIES  IN  PETRI  DISH  CULTURES    57 

possible  with  that  of  one  of  the  circles  on  the  counting 
plate. 

3.  Using  the  tripod  lens  count  the  colonies  in  each  sector 
of  the  smallest   circle,  then  in  each  division  between  the 
concentric  circles. 

Note  1.  The  tripod  counting  lens  must  be  used  if  the  colonies 
are  very  small,  as  they/ otherwise  may  be  confused  with  air  bubbles 
in  the  medium.  If  there  are  less  than  500  colonies  present,  the 
entire  plate  should  be  counted.  If  the  number  is  much  greater, 
from  ten  to  twenty  divisions,  in  some  definite  order,  should  be 
counted,  an  average  taken,  and  the  results  multiplied  by  the  area 
of  the  plate  in  square  centimeters. 

Note  2.  Wolfhugel's  counting  plate  is  very  desirable  for 
counting  a  large  number  of  colonies.  It  is  ruled  in  square  centi- 
meters and  the  squares  on  the  diagonals  of  the  plates  are  sub- 
divided into  smaller  squares.  The  colonies  appearing  in  from  ten 
to  twenty  of  these  smaller  squares  may  be.  counted,  an  average 
taken  and  the  result  multiplied  by  the  number  of  small  squares  in 
1  sq.  cm.  times  the  area  of  the  Petri  dish  in  square  centimeters. 
(The  entire  area  of  the  plate  may  be  obtained  most  quickly  by 
placing  a  Jeffer  plate  upon  the  Petri  dish  in  question.) 

4.  Ascertain  the  number  of  colonies  per  cubic  centi- 
meter in  the  original  sample  by  multiplying  the  whole  num- 
ber of  colonies  on  the  plate  by  the  dilution;   e.g.,  if  there 
are  386  colonies  on  the  plate  and  the  original  culture  was 
diluted  1  in  1000,  the  number  of  colonies  contained  in  each 
cubic  centimeter  of  the  original  sample  is  386,000. 

Note.  When  there  is  an  excessive  number  of  colonies  on  a  plate 
the  vigorous  microorganisms  will  inhibit  the  growth  of  the  less  vigorous 
and  thus  the  number  of  colonies  counted  is  smaller  than  the  number 
of  microorganisms  present.  Moreover,  the  colonies  may  become 
confluent  and  the  counts  will  again  be  in  error. 


58  GENERAL  MICROBIOLOGY 


EXERCISE  15.  ISOLATION  OF  MICROORGANISMS 
FROM  PLATE  CULTURES  AND  METHOD  OF 
MAKING  AGAR  STREAK  CULTURE 

Apparatus.  Straight  platinum  needle;  several  tubes 
of  sterile  agar  slants;  Bunsen  burner;  wax  pencil;  plate 
containing  from  30  to  200  well-separated  colonies. 

Method.  1.  Examine  the  plate  to  determine  the  colo- 
nies which  differ  macroscopically  and  microscopically,  (Use 


FIG.  19. — Various  Forms  of  Platinum  Needles.     (Orig.  Northrup.) 

a  counting  lens  or  the  lowest  power  of  a  compound  micro- 
scope.) 

2.  Note  the  most  isolated  of  each  kind,  and  mark  them 
with  the  wax  pencil  upon  the  bottom  of  the  plate  to  insure 
picking  up  the  proper  colonies  later.     Also  note  how  the 
deep  and  surface  colonies  differ. 

3.  Examine  each  marked  colony  under  the  lowest  power 
of  the  microscope  to  make  sure  of  its  purity.     If  the  colony 
does  not  appear  to  be  wholly  isolated,  pick  up  a  small 
portion  of  it  with  a  sterile  platinum  needle  and  stain  with 
one  of  the  common  stains  (see  p.  88)  or  examine  it  in  the 


ISOLATION  OF  MICROORGANISMS 


59 


hanging  drop  (see  p.  76)  to  determine  if  more  than  one 
kind  of  organism  is  present. 

4.  If  the  colony  is  pure,  pick  up  a  portion  with  the 


o 

i 


sterile  needle,  or,  in  case  of  extremely  small  colonies,  remove 
the  cover  from  the  plate,  focus  the  low  power  of  the  micro- 
scope on  the  desired  colony  and  while  looking  through  the 
microscope,  fish  the  colony. 


60  GENERAL  MICROBIOLOGY 

5.  Transfer  to  one  of  the  agar  slants,  making  a  streak 
along  the  median  line  of  the  inclined  surface  of  the  agar, 
drawing  the  needle  from  the  base  to  the  top  of  the  slant. 

Note.  For  investigational  purposes,  when  dealing  with  unknown 
microorganisms,  the  following  method  is  more  accurate  for  obtaining 
them  in  pure  culture:  Transfer  to  a  tube  of  broth;  incubate  for 
twenty-four  hours  and  plate  a  second  time.  Isolate  from  the  twenty- 
four-hour  plate  culture. 

6.  Incubate  at  the  optimum  temperature. 

Note.  If  the  agar  slants  haye  become  dried  out  to  any  extent,  it 
is  necessary  that  the  agar  be  melted  and  re-slanted  in  order  that 
optimum  growth  may  take  place. 

EXERCISE  16.     METHOD  OF  MAKING  TRANSFERS  OF 
PURE  CULTURES  INTO  A  LIQUID  MEDIUM 

Directions  for  making  transfers  of  pure  cultures  from 
one  medium  to  another  must  be  followed  very  carefully, 
otherwise  extraneous  microorganisms  may  enter  and  hope- 
less confusion  result. 

Apparatus.  Test  tubes  containing  a  sterile  liquid 
nutrient  medium;  platinum  needle;  Bunsen  burner. 

Culture.     Pure  culture. 

Method.  1.  Flame  the  cotton  plugs  of  the  test  tubes 
containing  the  pure  culture  and  the  sterile  liquid  nutrient 
medium. 

2.  Sterilize  the  platinum  needle  in  the  flame. 

3.  Permit  it  to  cool  (about  one  minute  is  required). 

4.  Hold  it  in  the  right  hand  and  remove  the  cotton  plug 
of  the  culture  tube  with  the  little  finger  of  the  same  hand. 

5.  Take  up  a  very  little  of  the  culture  with  the  needle. 

6.  Replace  the  plug  of  the  culture  tube. 

7.  Remove  the  plug  of  the  tube  of  sterile  liquid  medium 
in  the  same  manner. 

8.  Insert  the  infected  needle  into  the  liquid. 

9.  Replace  the  plug. 

10.  Sterilize  the  needle  before  laying  it  down. 


METHOD   OF  MAKING  STAB  CULTURES  61 

EXERCISE  17.     METHOD  OF  MAKING  STAB  CULTURES 

Apparatus.  Tubes  of  sterile  agar  or  gelatin;  straight 
platinum. needle;  Bunsen  burner. 

Culture.     Pure  culture. 

Method.  1.  Liquefy  the  gelatin  or  agar  tube  and  re- 
solidify it  in  a  vertical  position  in  cold  running  water  or 
in  some  cold  place. 

2.  With  a  sterilized  straight  platinum  needle  pick  up  a 
very  little  of  the  culture  or  colony. 

3.  Insert  the  needle  at  the  middle  of  the  circle  made  by 
the  surface  of  the  medium  and  push  the   needle  about 
5  cms.  into  the  solid  medium  (within  1  cm.  of  the  bottom 
of  the  tube),  then  withdraw  carefully  so  that  the  path  of 
the  needle  be  as  limited  as  possible.     The  microorganisms 
grow  along  the  path  of  the  needle. 

Avoid  having  the  shoulder  of  the  rod  come  in  contact 
with  the  surface  of  the  medium  lest  its  heat  disfigure  the 
surface  or  even  kill  the  microorganisms.  The  surface  of 
the  medium  should  remain  intact  during  this  process. 

4.  Replace  the  plug  in   the   new  culture  and  sterilize 
the  needle. 

EXERCISE  13.     PREPARATION  OF  A  GIANT  COLONY 

Purpose.  To  show  the  development  of  a  single  colony 
of  a  microorganism. 

Apparatus.  Sterile  Roux  culture  flask*  or  Petri  dish; 
tubes  of  agar  or  gelatin. 

Culture.     Organism  to  be  studied. 

Method.     1.  Melt  two  tubes  of  dextrose  agar  or  gelatin. 

Pour  into  the  culture  flask. 

Note.     Allow  the  medium  to  touch  and  cover  one  large  side  only. 

2.  Heat  in   this   horizontal   position  in   flowing  steam 
fifteen  minutes. 

3.  Distribute  the  medium  evenly  over  the  large  side, 
*  Most  valuable  for  molds,  especially  Rhizopus  nigricans. 


62 


GENERAL  MICROBIOLOGY 


and  set  on  a  level  surface  to  cool.     When  the  medium  has 
solidified,  place  the  flask  with  the  medium-side  up. 

4.  Mark  the  center  of  the  flask  on  the  outside  with  a 


FIG.  21.— Giant  Colony  of  Mold  in  Roux  Flask.     (Orig.) 

wax  pencil  and  inoculate  with  a  bent  platinum  needle  in 
one  spot  only. 

Note.  When  making  mold  inoculations  moisten  the  sterile  needle 
with  sterile  water  or  medium  before  touching  the  spores;  this  insures  a 
positive  inoculation. 

5.  Keep  at  room  temperature  medium-side  up. 

6.  Examine   and   measure   the   diameter   of   the   giant 
colony  from  day  to  day  and  describe  the  typical  growth 


THE  MICROSCOPE  63 

of  the  colony,  using  the  terms  on  the  descriptive  chart 
of  the  Society  of  American  Bacteriologists,  p.  134,  as  far 
as  possible. 

7.  Compare  the  giant  colonies  of  a  Mucor,  Pencillium, 
a  yeast  and  Bacillus  subtilis  or  Bacillus  mycoides.     Use 
agar  for  giant  colonies  of  these  bacteria,  as  they  liquefy 
gelatin. 

8.  Giant  colonies  of  yeasts  and  bacteria  and  some  molds 
may  be  grown  in  Petri  dishes,  or  in  flat-bottomed  flasks. 

For  illustrations  of  giant  colonies  of  bacteria  see: 

FUHRMAN:  Vorlesungen  iiber  Technische  Mykologie,  pp.  41,  43. 
LOHNIS:    Vorlesungen  iiber  Landwirtschaftliche  Bakteriologie,  pp.  38, 

170. 
LEHMANN  AND  NEUMANN:  Bakteriologie  und  Bakteriologische  Diag- 

nostik,  Bd.  I.     (Atlas.) 

For  illustrations  of  giant  colonies  of  yeasts  see: 

LAFAR:  Technische  Mykologie,  Bd.  4,  German  Ed.,   pp.  24-25,  306, 
and  above  references. 

THE   MICROSCOPE 

Care  of  the  Microscope.  For  microbiological  work  a 
compound  microscope  is  necessary.  This  should  be  fitted 
with  a  minimum  of  two  oculars  corresponding  to  the  Leitz 
No.  1  (lowest  power)  (Spencer,  6X),  and  No.  3  or  4  (Spencer, 
10  X)  and  three  objectives  corresponding  to  the  Leitz 
J  in.  (lowest  power)  (Spencer,  16  mm.),  -f  in.  (Spencer,  4 
mm.)  objectives  (dry)  and  ^  in.  oil  immersion  objective. 
A  coarse  and  a  fine  adjustment  permit  the  accurate  focus- 
ing of  any  combination  of  lenses.  The  substage  shoi  Id  be 
fitted  with  a  good  condenser  and  iris  diaphragm  for  regulating 
the  amount  of  light,  and  a  plane-concave  mirror. 

Great  care  should  be  exercised  in  the  use  and  care  of  the 
microscope  as  it  is  a  delicately  adjusted  instrument. 

The  following  rules  should  be  heeded: 

The  Stand.  The  stand  is  the  body  of  the  microscope 
carrying  the  optical  parts. 


64 


GENERAL  MICROBIOLOGY 


FIG.  22. — Compound  Microscope  with  Mechanical  Stage  Attached  and 
Side  Fine  Adjustment. 


THE  MICROSCOPE  65 

1.  Leave  the  microscope  in  the  case  when  not  in  use.     Dust 
works  into  the  bearings  of  the   instrument,  making  them 
work  hard  and  unnecessarily  wearing  them. 

2.  When  handling  the  microscope  do  not  grasp  it  by  the 
arm  which  contains  the  fine  adjustment  unless  the  micro- 
scope is  designed  to  permit  this.     Grasp  it  by  the  pillar 
below  the  stage. 

3.  Never  use  alcohol  on  the  lacquered  parts.     Rubbing 
gently  with   a  very  little  xylol   and   drying  quickly  will 
remove  any  oily  material. 

The  Stage.  The  stage  is  that  portion  of  the  microscope 
on  which  the  mounted  object  is  placed  for  examination. 

1.  Should  the  stage  become  soiled  with  balsam,  immer- 
sion oil  or  anything  which  water  will  not  remove,  it  can  be 
cleaned  with  xylol  or  chloroform.  A  little  heavy  oil  will 
restore  the  stage  to  its  original  black  color. 

The  Fine  Adjustment.  The  fine  adjustment  is  used  for 
bringing  out  details  in  very  small  objects  and  is  necessarily 
of  limited  range  and  delicate  in  its  mechanism. 

1.  If,  when  looking  into  the  eye-piece,  no  change  of  focus 
is  noticed  by  turning  the  micrometer  head,  or  if  the  microm- 
eter head  ceases  to  turn,  the  adjustment  has  reached  its 
limit.     To  adjust,  focus  down  or  up,  respectively,  with  the 
coarse  adjustment,   and  turn  the  micrometer  head  until 
the  fine  adjustment  is  midway  within  its  range. 

2.  When  the  fine  adjustment  screw  stops,  do  not  force  it. 
The  Draw  Tube.     The  draw  tube  is  the  tube  receiving 

the  ocular. 

1.  The  draw  tube  should  work  easily  and  smoothly. 
On  the  draw  tube  will  be  found  graduations  in  millimeters 
or  inches,  some  fixed  point  at  which  certain  combinations 
of  objectives  and  oculars  give  the  clearest  image.  This 
differs  with  different  microscopes  and  should  be  known  for 
the  microscope  used. 

The  Nose-piece.  The  triple  nose-piece  on  the  compound 
microscope  serves  a  double  purpose;  to  obviate  the  neces- 


66  GENERAL  MICROBIOLOGY 

sity  of  screwing  the  different  objectives  in  as  needed  and 
to  protect  the  back  lens  of  the  objective  from  dust.  The 
later  microscopes  have  a  "  collar  "  nose-piece  which  keeps 
the  objectives  free  from  dust  at  all  times. 

1.  Nose-pieces  and  objectives  of  the  best  makes  are  now 
made  so  that  the  objectives  are  parfocal,  i.e.,  when  one  lens 
is  in. focus  the  others  on  the  nose-piece  will  be  nearly  in 
focus  when  they  are  swung  into  the  optical  axis.     They 
are  also  approximately  centered  so  that  a  point  in  the 
center  of  the  field  of  one  lens  will  be  in  the  field  of  the 
others. 

2.  Objectives  made  parfocal  for  one  tube-length  or  eye- 
piece are  not  parfocal  for  a  different  length  or  a  different 
eye-piece. 

3.  Objectives  of  one  microscope  should  not  be  inter- 
changed with  those  of  another,  even  if  of  the  same  make. 

4.  Always  focus  up,  slightly,  before  turning  from  a  lower 
to  a  higher  power.     Otherwise  the  front  of  the  objective 
may  be  swung  against  the  cover-glass  and  injure  both  the 
specimen  and  the  objective. 

The  Optical  Parts.     The  optical  parts  are  the  lenses  of 
the  objectives,  oculars  and  condenser  and  the  mirror. 

1.  Wipe  dirty  lenses  gently  with  Japanese  lens  paper  to 
remove  dirt. 

2.  Never  rub  a  lens  vigorously  with  anything. 

3.  Avoid  touching  the  surface  of  a  lens  with  the  fingers. 
Cutaneous  secretions  are  hard  to  remove. 

4.  Always   clean  the  oil  immersion  objective  with   lens 
paper  immediately  after  using.     If  the  oil  is  allowed  to  dry, 
xylol  must  be  used  to  clean  the  lens. 

5.  Always  leave  an  ocular  in  the  tube  to  keep  dust  from 
settling  on  the  back  lens  of  the  objective.     Dust  on  the 
back  lens  may  be  removed  with  a  earners  hair  brush. 

6.  Never  take  an  objective  apart. 

7.  Oculars,  condenser  and  mirror  should  be  kept  clean 
by  the  use  of  lens  paper. 


THE  MICROSCOPE 


67 


Use  of  the  Microscope.  Position.  1.  Always  use  the 
microscope  with  the  tube  in  the  perpendicular  position. 
This  is  indispensable  in  examining  fresh  mounts  or  fluids. 

2.  Work  with  both  eyes  open  and  if  possible  use  both 
eyes  interchangeably. 

Light.  3.  Never  use  direct  sunlight.  The  best  light  is 
obtained  from  white  clouds.  Northern  or  eastern  light  is 
preferable. 

The  best  artificial  light  is  a  Welsbach  burner  (gas). 
When  employing  artificial  light  use  a  blue  glass  between 


FIG.  23. — Double  Demonstration  Ocular  with  Pointer  Enabling  Two 
Observers  to  View  Simultaneously  the  Image  Indicated  by  the 
Adjustable  Pointer. 

the  light  source  and  the  specimen.  Often  an  eye-shade 
or  some  appliance  with  a  similar  purpose  is  desirable. 

4.  Use  the  plane  mirror  in  daylight,  the  concave  mirror 
with  artificial  light. 

Focusing.  5.  After  putting  in  place  a  low-power  ocular 
and  objective,  place  the  specimen  on  the  stage,  and  while 
looking  through  the  microscope,  adjust  the  mirror  so  as  to 
illuminate  the  field  as  evenly  as  possible,  but  not  so  brightly 
as  to  irritate  the  eyes. 

6.  By  means  of  the  coarse  adjustment,  focus  the  body 
tube  until  the  objective  nearly  touches  the  cover-glass, 
being  careful  not  to  touch  it. 


68 


GENERAL  MICROBIOLOGY 


7.  With  the  eye  at  the  ocular,  focus  up  slowly  with  the 
coarse  adjustment  until  the  specimen  comes  plainly  into 
view. 

//  the  light  is  too  intense  the  focal  point  may  be  passed 
without  noticing  it. 

8.  When  the  object  is  brought  fairly  well  into  focus  by 


FIG.  24. — Comparison  Ocular  Enabling  One  to  Observe  Two  Different 
Microscopic  Fields  Side  by  Side.  Any  two  microscopes  may 
be  used,  (Healy.) 

means  of  the  coarse  adjustment,  use  the  fine  adjustment 
to  obtain  the  sharpest  focus  to  bring  out  details. 

9.  Move  the  specimen  when  trying  to  obtain  a  focus, 
as  a  moving  object  is  more  apt  to  be  noticed  as  the  lens 
comes  into  focus. 

The  microscope  reverses  the  image.  This  will  be  noticed 
when  the  specimen  is  moved.  The  microscope  magnifies 
the  movement  as  well  as  the  image;  it  therefore  requires 


THE  MICROSCOPE  69 

a  certain  delicacy  of  movement  to  put  a  specimen  in  a 
desired  position. 

10.  Beginners  should  always  use  the  low-power  objectives 
and  oculars  first.     The  low-power  objectives  have  longer 
working  distances  and  always  show  a  larger  portion  of  the 
specimen.     After  obtaining  a  general  idea  of  the  specimen, 
desired  portions  may  be  examined  with  the  higher  power 
objectives. 

11.  In  using  high-power  objectives  for  finding  and  exam- 
ining a  specimen,  it  is  always  more  desirable  to  use  the  low- 
est power  ocular  (corresponding  to  Leitz  No.  1).     If  a  higher 
ocular  is  used,  there  is  a  loss  in  the  depth  or  sharpness  and 
size  of  field,  since  they  are  both  inversely  proportional  to 
the  magnification.     Illumination  is  also  lost,  which  varies 
inversely  as  the  square  of  the  magnification.     Remember 
that  the  largest  field,  the  greatest  penetration,  and  the  best 
illumination  are  obtained  by  using  the  lowest  magnification 
which  makes  all  the  detail  in  the  image  visible. 

Oil  Immersion  Objective.  The  highest  power  objective 
is  the  oil  immersion  lens.  This  is  so  termed  because  a  drop 
of  oil  must  be  used  between  the  front  lens  and  the  cover- 
glass.  The  oil  used  must  have  the  same  index  of  refrac- 
tion as  glass  to  prevent  the  dispersion  of  the  rays  of  light 
coming  from  the  condenser. 

Working  distance  is  the  free  distance  between  the  cover- 
glass  and  the  objective  when  the  latter  is  focused.  High- 
power  objectives  have  short  working  distances. 

REFERENCE 

GAGE:  The  Microscope. 


70  GENERAL  MICROBIOLOGY 


EXERCISE  19.     METHOD  OF  MEASURING  MICRO- 
ORGANISMS 

1.  Using  the   Leitz   Ocular   "  Step  "   Micrometer.     In 
this  ocular  micrometer  the  intervals  are  arranged  in  groups 
of  ten,  each  group  being  indicated  by  black  steps  rising 
from  the  first  to  the  tenth  interval. 

This  arrangement  possesses  the  great  advantage  that  the 
divisions  can  always  be  seen  distinctly  whether  the  objects 
be  light  or  comparatively  dark. 

The  intervals  of  the  scale,  instead  of  being  0.1  mm.  or 
0.5  mm.  wide,  as  in  ordinary  ocular  micrometers,  have  a 
definite  value  of  0.06  mm.  This  gives  for  each  objective 
and  for  a  given  tube  length,  convenient  and  in  many  cases 
integral  micrometer  values,  which  renders  a  greater  facil- 
ity in  the  use  of  this  instrument.  The  actual  tube  length 
differs  in  most  cases  but  little  from  the  standard  length. 
The  tube  length  and  the  micrometer  value  of  each  micro- 
scope, however,  should  be  separately  calibrated. 

It  is  of  importance  to  be  able  to  determine  the  size  of 
microorganisms:  (1)  because  it  is  of  general  interest  to 
know  the  size  of  the  microorganisms  with  which  we  are 
dealing;  (2)  because  the  difference  in  size  is  an  important 
factor  in'  identifying  and  describing  the  organism;  (3) 
because  the  size  is  necessary  for  purposes  of  comparison 
with  other  microorganisms. 

Apparatus.  Microscope;  Leitz  ocular  "  step  "  microm- 
eter; object  micrometer;  specimen  to  be  measured. 

Method.  1.  With  the  aid  of  the  Leitz  ocular  "  step  " 
micrometer  the  size  of  stained  or  unstained  microorganisms 
on  either  a  light  or  a  dark  field  may  be  measured  directly 
in  microns. 

A  micron  is  0.001  mm.,  and  is  expressed  by  the  Greek 
letter  p.. 

2.  One  hundred  divisions  of  the  step  micrometer  cover 
100,     15    and    10    divisions    of    the    object    micrometer 


METHOD  OF  MEASURING  MICROORGANISMS        71 


when    Leitz  objectives  3,   7  and  1/12    oil  immersion  are 
used. 

The  object  micrometer  is  simply  a 
cover-glass  (mounted  on  a  slide  in 
Canada  balsam)  upon  whose  surface 
has  been  ruled  a  scale  2  mm.  in  length, 
each  millimeter  being  divided  into  100 
equal  parts,  the  space  between  each  di- 
vision therefore  being  equal  to  0.01  mm. 

3.  If     100    division    lines    of    the 
ocular  step  micrometer  cover  0.01  mm. 
of   the   object  micrometer,   then  each 
division  line  of  the  step  micrometer  has 
the  value  0.0001  mm.  or  0.1  micron. 

These  values  are  only  accurate  when 
the  draw-tube  of  the  microscope  is 
drawn  out  according  to  the  following 
table. 

4.  Using  the  ocular  step  micrometer 
and  the  object  micrometer,  find  the  tube 
length  at  which  each   objective   gives   a 
definite  value  in  microns.     This  will  vary 
some  even  with  the  Leitz  oculars  and 

objectives,  so  the  tube  length  for  each    FIG.  25.— Micrometer 
combination  of  lenses  must  be  deter- 
mined   separately   for    any    make    of 
microscope. 


-:JO 


40 


70 


•80 


Scale  in  Ocular  of 
the  Leitz  Ocular 
Step  Micrometer. 


VALUES   FOR   LEITZ    ACHROMATIC    OBJECTIVES 


No.  of  Objective. 

Mark  on  Draw-tube. 

Micrometer  Value  in  Microns  of  Each 
Division  Line  of  Step  Micrometer. 

3 

7 
1/12  oil  imm. 

141 
174 
150 

10 
1.5 
1.0 

5.  Multiply  the  number  of  division  lines  of  the  ocular 
micrometer  covered  by  the  organism  in  question  by  the  value 


72 


GENERAL  MICROBIOLOGY 


of  the  division  line  as  determined  in  the  above  table.     This 
gives  the  measurement  directly  in  microns. 

Microorganisms  may  be  measured  more  accurately  by 
mounting  them  in  Chinese  ink,  as  they  cannot  move,  are 
not  shrunken  or  distorted  as  often  occurs  with  stained  speci- 
mens, and  are  clearly  seen.  Preparations  stained  with 


FIG.  26. — Ocular  Filar  Micrometer  for  Very  Exact  Measurements. 
By  means  of  a  micrometer  screw  a  line  is  moved  across  the 
field.  The  distance  is  measured  by  means  of  the  divisions  on 
the  drum. 

aqueous-alcoholic  dyes  stand  next  in  preference,  never 
strong  stains  like  carbol-fuchsin,  anilin-water  dyes,  or 
saturated  alcoholic  solutions  of  dyes. 

II.  Using  a  Filar  Ocular  Micrometer.  The  filar  ocular 
micrometer  is  an  instrument  for  the  accurate  measure- 
ment of  microscopic  objects.  It  consists  of  an  ocular, 
between  the  eye  and  field  lenses  of  which  there  is  a  scale 
ruled  on  glass  in  millimeters  and  half  millimeters,  below 


METHOD  OF  MEASURING  MICROORGANISMS        73 

and  across  which  a  single-line  index  is  made  to  travel  by  the 
use  of  the  micrometer  screw. 

The  micrometer  screw  is  fitted  with  a  drum  divided  into 
100  parts,  one  revolution  of  which  moves  the  index  line 
one  division  or  5  microns.  The  drum  is  divided  into  fifty 
parts,  so  that  each  mark  on  the  drum  scale  corresponds  to 

5  microns 

or  0.1  micron. 

oU 

The  micrometer  value  of  each  interval  should  be  cali- 
brated for  each  objective  with  the  aid  of  the  object  microm- 
eter. The  eye-lens  of  the  micrometer  is  adjustable  to  enable 
the  observer  to  focus  the  scale  accurately. 

The  filar  ocular  micrometer  slips  into  the  draw-tube 
of  the  microscope  like  any  ordinary  ocular  and  may  be 
fixed  in  position  by  the  milled-head  screw  on  the  side. 

A.  Calibration  of  the  Filar  Ocular  Micrometer. 

Apparatus.  Filar  ocular  micrometer;  microscope;  ob- 
ject micrometer. 

Method.  1.  Place  the  object  micrometer  under  object- 
ive No.  3  and  ocular  No.  1,  drawing  out  the  draw-tube  to 
17  mm. 

2.  Bring  the  lines  on  the  object  micrometer  into  a  sharp 
focus. 

3.  Replace  ocular  No.   1  with  the  filar  ocular  microm- 
eter. 

4.  Focus  again  so  that  the  division  of  the  object  microm- 
eter and  the  ocular  micrometer  are  equally  clear  and  turn 
the  ocular  micrometer  so  that  the  lines  of  both  micrometers 
are  parallel  to  each  other. 

5.  Determine  how  many  microns  one  space  of  the  ocular 
micrometer  represents. 

Example.  Six  divisions  of  the  ocular  micrometer-scale  cover  the 
same  length  as  three  divisions  of  the  object  micrometer  on  which  each 
division  is  1/100  millimeter  or  10  microns;  therefore,  six  divisions  of 
the  ocular  micrometer  scale  equals  30  microns  and  one  division  equals 
1/6  of  30  or  5  microns. 


74  GENERAL  MICROBIOLOGY 

6.  Determine  how  many  revolutions  of  the  drum  (from 
0  to  0)  are  necessary  to  move  the  movable  line  one  division 
and  from  this  determination  calculate  the  value  in  microns, 
of  one  division  on  the  drum.  One  determination  of  this 
value  is  sufficient. 

B.  Method  of  Using  the  Filar  Ocular  Micrometer. 

1.  Replace  the  object  micrometer  by  a  slide  containing 
organisms,  focus,  and  measure  an  organism,  counting  the 
number  of  divisions  the  drum  is  turned  in  moving  the 
movable  line  from  end  to  end  of  the  organism. 

Example.  If  the  drum  is  turned  two  divisions  the  organism  was 
two  times  0.1  micron  in  length  or  0.2  micron. 

2.  To  measure  the  microorganisms  with  a  higher  power 
objective,  the  value  of  each  division  of  the  scale  has  to  be 
recalibrated. 

EXERCISE    20.     DETERMINATION    OF   THE    RATE    OF 
MOVEMENT  OF  MOTILE  ORGANISMS 

Apparatus.  Microscope;  Leitz  "step"  micrometer;  stop- 
watch; hanging-drop  preparation  of  motile  organisms. 

Method.  Using  a  hanging-drop  preparation  of  the 
organism  to  be  examined,  determine  the  rate  of  movement 
per  second,  using  the  step  micrometer  and  a  stop-watch. 

EXERCISE  21.     PREPARATION   OF  A  HANGING  DROP 

The  purpose  of  the  hanging-drop  preparation  is  to  study 
bacteria  in  the  living  condition;  to  demonstrate  (a)  their 
form,  (6)  arrangement,  (c)  motility  (this  is  best  ob- 
served from  twenty-four-hour  cultures),  (d)  appearance, 
(e)  division  of  cells,  (/)  formation  or  presence  of  spores; 
(g)  to  determine  the  presence  and  types^of  microorganisms  in 
any  material  and  to  watch  the  changes  in  the  predominat- 
ing types  of  the  microbial  flora  in  a  medium  from  day  to 
day;  (h)  and,  in  pathogenic  bacteriology,  to  demonstrate 
agglutination. 


PREPARATION  OF  A  HANGING  DROP 


75 


Bacteria  have  two  kinds  of  movement,  the  so-called 
Brownian  or  molecular  movement,  and  true  motility.  The 
former  may  be  demonstrated  by  examining  the  movement 
of  powdered  carmen  rubrum  in  the  hanging  drop.  A  very 
little  of  the  powder  is  sufficient.  Brownian  movement 
is  shown  more  or  less  by  all  small  particles  of  insoluble 
matter  (including  living  non-motile  or  dead  bacteria)  in 
suspension.  It  is  characterized  by  a  vibratory  movement 
affecting  the  entire  field;  the  relative  positions  of  the 
insoluble  particles  are  never  altered.  This  type  of  move- 


FIG.  27. — Hanging  Drop  Slide.     (Orig.) 


ment  must  be  distinguished  from  that  of  true  motility, 
which  is  characterized  by  the  progressive  movement,  more 
or  less  rapid,  of  an  organism  across  the  field  of  the  micro- 
scope, changing  its  position  in  the  field  independently  of 
and  in  a  direction  contrary  to  other  organisms  present. 

There  should  be  no  currents  of  air  entering  under  the 
cover-glass  and  passing  through  the  concavity  of  the  slide 
nor  should  there  be  currents  in  the  liquid.  The  latter  may 
occur  if  the  organisms  have  not  been  well  mixed  through 
the  drop  in  the  process  of  preparation.  If  large  numbers 
of  the  microorganisms  in  the  drop  are  moving  in  one  direc- 


76  GENERAL  MICROBIOLOGY 

tion,  this  is  an  indication  of  currents  in  the  liquid  which 
have  been  induced  by  the  liquid  touching  the  side  of  the 
concavity,  by  the  drop  being  too  large,  by  improper  mixing, 
or  by  air  currents;  this  fault  may  be  remedied  by  thoroughly 
mixing  the  bacteria  in  the  drop  with  the  straight  needle 
or  by  resealing  the  cover-glass  upon  the  slide. 

Apparatus.  Clean  cover-glasses;  clean  concave  slides; 
platinum  loop;  straight  platinum  needle;  Bunsen  burner; 
distilled  water;  cover-glass  forceps;  melted  paraffin  or 
vaselin  if  preparation  is  to  be  sealed  permanently. 

Method.  1.  With  a  platinum  loop  place  four  small 
drops  of  water  about  the  edge  of  the  depression  of  the  con- 
cave slide. 

2.  For  cultures: 

In  liquid  media. 

(a)  With  a  sterile  platinum  loop  transfer  a  portion 
of  the  culture  to  the  center  of  a  clean  cover- 
glass. 

On  solid  media. 

(a)  With  a  sterile  platinum  loop  place  a  small  drop 
of  water  or  physiological  salt  solution  in  the 
center  of  a  clean  cover-glass. 

(6)  With  a  sterile  platinum  needle  transfer  a  minute 
portion  of  the  culture  to  the  drop  of  water  so 
that  only  the  faintest  cloudiness  appears. 

3.  Quickly  invert  the  cover-glass  over  the  depression  in 
the  concave  slide  and  gently  depress  the  margin  on  the  water 
until  the  chamber  is  sealed  air  tight.     The  hanging  drop 
must  not  touch  the  bottom  of  the  concavity.     Note  the 
illustration.     If  it  is   desired   to   keep   the   hanging   drop 
longer  than  five  to  ten  minutes,  it  may  be  sealed  with 
paraffin  as  with  the  adhesion  culture,  or  with  vaselin. 

The  drop  must  remain  over  the  center  of  the  concavity. 
If  the  drop  touches  the  side  of  the  concavity,  the  hanging 
drop  as  such  is  destroyed  and  it  will  be  necessary  to  remake 
the  preparation.  If  pathogenic  organisms  are  used,  both 


PKEPARATION  OF  A  HANGING  DROP  77 

slide  and  cover-glass  must  be  placed  in  1/1000  mercuric 
chloride  or  some  equally  efficient  disinfectant  for  at  least 
one  hour  before  cleaning  or  reusing. 

4.  Examine  first  with  objective  No.  3,  then  with  object- 
ive No.  7  or  the  1/12  oil  immersion  lens,  using  ocular  No.  1 
in  each  case.  After  a  perfect  focus  is  obtained,  ocular 
No.  4  may  be  used  if  desired. 

Manipulation  of  Microscope.  Using  the  lowest  power 
objective  and  ocular,  focus  the  tube  of  the  microscope 
down  by  means  of  the  coarse  adjustment  until  the  objective 
nearly  touches  the  cover-glass,  being  careful  not  to  touch  it. 
Then,  with  the  eye  at  the  ocular,  focus  up  with  the  coarse 
adjustment  and  move  the  preparation  until  the  edge  of  the 
drop  comes  plainly  into  view.  This  focal  point  may  be  passed 
without  noticing  it  if  the  light  is  too  intense  or  too  dim.  The 
edge  of  the  drop  is  a  curved  line.  The  preparation 
should  be  so  moved  that  this  line  cuts  the  center  of  the 
field. 

Focus  up  slightly,  swing  the  No.  7  or  1/12  objective  as 
desired,  into  place  and  after  the  field  desired  is  obtained 
with  the  coarse  adjustment,  focus  down  until  the  objective 
nearly  touches  the  cover-glass.  Then  with  the  eye  at  the 
ocular,  focus  up  carefully  with  the  coarse  adjustment  until 
the  edge  of  the  drop  comes  plainly  into  view.  Use  the  fine 
adjustment  to  bring  out  details. 

In  using  the  1/12  oil  immersion  lens  a  small  drop  of  im- 
mersion oil  is  placed  in  the  center  of  the  cover-glass,  the 
1/12  objective  swung  into  place  as  above.  Greater  care 
must  be  exercised  in  focusing,  as  this  objective  has  a  shorter 
working  distance. 


78 


GENERAL  MICROBIOLOGY 


EXERCISE  22.     PREPARATION  OF  THE  ADHESION 
CULTURE 

The  purpose  of  the  exercise  is  to  show  the  germination 
of  mold  spores  or  the  budding  of  yeast  cells,  i.e.,  colony 
formation. 

Apparatus.  Clean  cover-glasses;  clean  concave  slides; 
melted  paraffin;  small  glass  rod  or  camel's-hair  brush; 


FIG. 


28. — Lindner's  Adhesion  Culture.     (Adapted  from   Lafar's 
Technische  Mykologie.) 


sterile  cider  in  tubes.  (Wort,  milk  and  other  media  may  be 
used  as  conditions  demand.) 

Cultures.  Pure  culture  of  mold,  or  yeast.  If  mold 
spores  are  to  be  germinated,  an  old  culture  having  spores  is 
necessary. 

Method.  1.  Inoculate  a  tube  of  sterile  cider  from  the 
pure  culture  of  the  organisms  to  be  studied.  (Use  spores 
in  the  case  of  mold  and  some  of  the  cells  for  yeasts.)  Dis- 
tribute well  with  the  platinum  needle. 

2.  Transfer  one  loopful  to  a  flamed  cover-glass  and 
spread  in  a  thin  film  over  the  entire  surface  of  the  cover- 
glass,  using  the  straight  needle.  If  any  of  the  cider  adheres 
in  droplets,  shake  them  off. 


PREPARATION   OF  THE  ADHESION  CULTURE       79 

3.  Breathe  into  the  concavity  of  a  concave  slide  until 
small  droplets  of  moisture  are  visible  on  the  glass.     Before 
this    moisture    evaporates    and    while    the    cover-glass    is 
still  wet  turn  the  cover-glass,  culture  side  down,  corner- 
wise,  covering  the  concavity  on  the  slide. 

4.  Using  the  small  glass  rod  or  a  camel's-hair    brush 
dipped  in  hot  paraffin,  neatly  seal  the  cover-glass  on  the 
slide  so  that  the  cavity  will  be  air  tight  and  the  moisture 
will  be  retained.     Success  depends  largely  on  quick  work. 

5.  Examine  with  objective  No.   7  and  ocular  No.    1. 
There  should  be  five  to  twenty  spores  or  cells  on  a  slide. 
If  more  are  found,   a  new   culture  should   be  made.     It 
may    be    necessary  to    inoculate  a  second    tube  of  cider 
from  the  first  to  secure  the  proper  dilution. 

6.  If  you  are  not  familiar  with  the  spores  or  cells  of 
the  organism  to  be  studied,   before  making  an  adhesion 
culture,  mount  them  in  a  drop  of  water  heavily  inoculated, 
cover  with  a  cover-glass  and  examine  microscopically. 

7.  Keep  the  cultures  at  room  temperature.     Examine 
as  often  as  possible  for  thirty-six  hours  and  then  every 
twenty-four  hours  till  growth  ceases. 

8.  Draw  as  many  stages  as  possible.     The  time  required 
for  spore  germination  is  usually  six  to  forty-eight  hours. 

Note.  Some  molds  grow  quite  extensively  in  the  adhesion  culture, 
even  producing  fruiting  bodies.  Very  often  both  the  mycelium  and  fruit- 
ing bodies  show  peculiar  abnormalities  and  should  never  be  drawn  to  repre- 
sent normal  structures.  These  abnormalities  are  the  result  of  the 
peculiar  environment. 

9.  Failure  to  obtain  growth  of  the  mold  spores  or  yeast 
cells  may  be  due  to  imperfect  sealing,  insufficient  moisture 
at  the  start  or  too  many  cells  on  the  cover-glass.     If  the 
adhesion  culture  fails  to  grow,  a  fresh  tube  of  cider  must 
be  inoculated  before  making  new  adhesion  cultures,  as  the 
food  materials  contained  in  the  medium  are  partly  or  en- 
tirely used.     In  the  case  of  mold  spores  it  is  reasonable 
to  expect  that  any  mold  spores  in  the  adhesion  culture 


80  GENERAL  MICROBIOLOGY 

will  have  germinated  within  forty-eight  hours  after  prepar- 
ing the  mount. 

Note.     This  method  may  be  utilized  to  study  the  colony  develop- 
ment of  bacteria  also. 

EXERCISE  23.     PREPARATION  OF  THE  MOIST- 
CHAMBER  CULTURE 

The  purpose  of  the  exercise  is  to  study  colony  formation 
in  molds,  yeasts  and  bacteria. 

Apparatus.     Clean    cover-glasses;     small    glass     rings, 


FIG.  29. — Moist  Chamber  Culture.     (Orig.) 

clean;  clean  slides;  sterile  pipette;  paraffin  or  vaselin; 
sterile  distilled  water  in  test  tube;  tube  of  a  sterile  liquid 
nutrient  medium;  platinum  needle  and  loop;  forceps; 
cover-glass. 

Culture.     Pure  culture  of  organism  to  be  studied. 

Method.  1.  With  forceps,  carefully  sterilize  in  a 
flame  a  glass  slide  and  a  glass  ring  designed  for  this  pur- 
pose. This  should  be  done  by  a  swinging  motion  to  insure 
uniform  distribution  of  the  heat. 

2.  Around  the  edges  of  the  ring,  after  it  has  cooled  suf- 
ficiently, place  a  little  vaselin,  while  the  ring  is  still  held 
in  sterile  forceps. 


AGAR  HANGING-BLOCK  CULTURE  81 

3.  Place  the  ring  on  the  slide  and  press  it  down  gently 
to  make  contact  complete.     The  vaselin  renders  the  cham- 
ber water  tight. 

4.  Seal  the  ring  to  the  slide  with  melted  paraffin  as  in 
the  adhesion  culture,  to  keep  it  from  slipping  around. 

5.  With   a   sterile   pipette   convey   into    this   chamber 
just  enough  (boiled)  water  to  cover  the  bottom. 

6.  Vaselin  the  upper  edge  of  the  ring. 

7.  Inoculate  lightly  the  tube  of  liquid  medium  with  the 
organism  to  be  studied.     Distribute  throughout  the  liquid 
with  the  needle. 

8.  Transfer  one  loopful  to  a  cover-glass. 

9.  Using  the  straight  needle,  spread  in  a  thin  film  over 
the  entire  surface  of  the  cover-glass.     If  any  of  the  liquid 
adheres  in  droplets,  shake  them  off. 

10.  Press  the  cover-glass,  medium  side  down,  upon  the 
upper  vaselined  edge  of  the  ring. 

11.  Seal  the  edge  of  the  cover-glass  to  the  glass  ring  in 
several  places  with   paraffin  to  prevent  it  from  slipping 
around. 

12.  Incubate  at  the  desired  temperature. 

This  possesses  some  advantages  over  the  adhesion  cul- 
ture, as  more  air  and  moisture  and  consequently  more  favor- 
able conditions  are  furnished  for  growth.  With  a  little 
more  delicate  manipulation  agar  or  gelatin  can  be  used 
in  place  of  the  liquid  medium. 

EXERCISE    24.     PREPARATION    OF    AGAR    HANGING- 
BLOCK   CULTURE 

This  method  was  devised  by  Hill  *  for  studying  to 
better  advantage  the  morphology  and  manner  of  multi- 
plication of  bacteria. 

Carry  out  this  procedure  in  a  special  plating  room  or 
chamber  if  possible,  to  avoid  contamination  from  air  cur- 
rents. 

*Hill,  Journal  of  Medical  Research,  Vol.  VII,  March,  1902,  p.  202. 


82  GENERAL  MICROBIOLOGY 

Apparatus.  Clean  cover-glasses;  clean  concave  slides; 
ordinary  slides,  clean;  tube  of  sterile  nutrient  agar  or 
gelatin;  paraffin;  two  sterile  Petri  dishes:  scalpel;  plati- 
num loop. 

Culture.     Pure  culture  of  the  organism  to  be  studied. 

Method.  1.  Liquefy  a  tube  of  nutrient  agar  or  gelatin, 
pour  it  into  a  sterile  Petri  dish  to  the  depth  of  about  4  mm. 
and  allow  it  to  harden. 

2.  With  the  flame-sterilized  scalpel,  cut  out  a  block  of 
agar  about  8  mm.  square. 

3.  Raise  the  agar  block  on  the  blade  of  the  scalpel  and 
transfer  it,  under  side  down,  to  the  center  of  a  sterile  slide. 

4.  With  a  sterile  platinum  loop,  spread  a  drop  of  the 
liquid   culture   (or  suspension  of  organisms  from   a  solid 
culture  medium)  over  the  upper  surface  of  the  agar  block 
as  if  making  a  cover-glass  film. 

5.  Place  the  slide  and  block  in  a  sterile  Petri  dish  and 
incubate  for  ten  minutes  at  37°  C.  to  dry  slightly. 

6.  With  sterile  forceps,  lower  a  clean,  dry,  sterile  cover- 
glass  carefully  on  the  inoculated  surface  of  the  agar  (avoid- 
ing air  bubbles),  so  as  to  leave  a  clear  margin  of  cover- 
glass  overlapping  the  agar  block. 

7.  Invert  the  preparation  and,  with  the  blade  of  the 
scalpel,  remove  the  slide  from  the  agar  block. 

8.  With  the  platinum  loop,  run  a  drop  or  two  of  melted 
agar  around  the  edges  of  the  block.     This  solidifies  at  once 
and  seals  the  block  to  the  cover-glass. 

9.  Sterilize  a  concave  slide. 

10.  Invert  the  cover-glass  with  the  block  attached  on 
the  concave  slide  and  seal  it  in  place,  firmly,  with  paraffin. 

11.  Observe  immediately  and  later  from  time  to  time 
with  ocular  No.  1  and  objective  No.  7  or  the  oil  immersion 
lens. 


LINDNER'S  CONCAVE-SLIDE  METHOD  83 


EXERCISE    25.     LINDNER'S  CONCAVE-SLIDE  METHOD 
FOR  DEMONSTRATING  FERMENTATION 

The  object  of  this  exercise  is  to  test  the  fermenting 
power  of  yeasts. 

Apparatus.  Three  clean  concave  slides;  three  clean 
cover-glasses;  sterile  filter  paper  (place  several  pieces  8  cm. 
square  in  a  Petri  dish  and  sterilize  in  the  hot  air);  three 
tubes  of  sterile  wort  or  cider;  three  sterile  1  c.c.  pipettes; 
forceps;  melted  paraffin;  platinum  needles;  Bunsen  burner. 

Cultures.  Saccharomyces  cerevisice;  Saccharomyces  apicu- 
latus;  Torula  rosea. 

Method.  The  following  procedure  is  to  be  used  for  each 
organism  to  be  tested: 

1.  Using  the  straight  needle,  inoculate  a  tube  of  wort 
with  Saccharomyces   cerevisioe    and   mix   well  through  the 
medium. 

2.  Sterilize  a  concave  slide  in  the  flame. 

3.  Using   a   sterile    pipette,    fill   the   concavity   of   the 
slide  until  the  liquid  "  rounds  up  "  over  the  concavity. 

4.  Holding  a  cover-glass  in  the  forceps,  sterilize  it  in 
the  flame. 

5.  Lay  the  cover-glass  on  the  end  of  the  slide  and  push 
it  over  with  the  forceps  until  the  cover-glass  covers  the 
concavity,  thus  sealing  in  the  inoculated  liquid.     There  must 
be  no  air  bubbles.     The  preparation  must  be  made  over  again 
if  this  occurs. 

6.  Remove  the  excess  liquid  with  sterile  filter  paper, 
using  forceps  to  hold  the  paper. 

7.  Seal  the  cover-glass  with  paraffin  as  with  the  adhesion 
culture. 

8.  Place  the  slides   in   a    horizontal   position   in   Petri 
dishes,  or  in  a  slide  box  as  convenient. 

9.  Incubate  at  25°  to  30°  for  twenty-four  hours.     Gas 
bubbles  will  be  formed  in  twenty-four  to  forty-eight  hours, 
if  any  fermentation  occurs. 


84  GENERAL  MICROBIOLOGY 

10.  Record  the  time  of  fermentation  and  the  relative 
fermentation   of   each   yeast   and    draw   conclusions   from 
your  results. 

11.  Do  your  results  coincide  with  those  in  the  refer- 
ences given? 

12.  State  in  detail  your  results  with  any  conclusions 
which  follow  from  them,  and  point  out  the  practical  applica- 
tions which  may  be  made. 

By  the  use  of  sugar  broth  in  place  of  wort,  this  method 
may  be  employed  for  bacteria  as  well. 

REFERENCES 

LAFAR:  Technical  Mycology,  Vol.  II,  Part  1,  pp.  113,  114,  and  Part  2, 
pp.  401-407,  430-436.  (Index  of  three  volumes  is  in  Vol.  II, 
Part  2.) 

GREEN:  Soluble  Ferments  and  Fermentation,  pp.  333-362. 

CONN:  Bacteria,  Yeasts  and  Molds,  pp.  56-99. 

EXERCISE  26.     LINDNER'S  DROPLET  CULTURE 

The  object  of  the  exercise  is  to  isolate  a  single  yeast 
cell  and  watch  its  development. 

Apparatus.     Sterile  cover-glass  (sterilize  in  flame);  con- 


FIG.  30. — Lindner's  Droplet  Culture.     (Adapted  from  Lafar's  Tech- 
nische  Mykologie.) 

cave  slide;  forceps;  sterile  toothpick  (sterilize  in  a  test 
tube  in  hot  air) ;  paraffin;  India  ink. 

Culture.     Pure  culture  of  some  yeast. 

Method.  1.  Inoculate  a  tube  of  cider  with  yeast. 
Distribute  the  organisms  well. 

2.  Using  the  sterile  toothpick,  make  five  rows  of  small 


CHINESE  INK  PREPARATION  85 

2.  Using  the  sterile  toothpick,  make  five  rows  of  small 
droplets  (five  droplets  in  a  row)  on  a  sterile  cover-glass  and 
place,  culture  side  down,  over  the  concavity  of  a  sterile  slide. 

3.  Seal  the  cover-glass  with  paraffin  as  in  the  prepara- 
tion of  the  adhesion  culture.     Examine  microscopically. 

4.  Locate   one   droplet   which   contains   only   one   cell. 
Using  India  ink,  write  the  location  of  this  droplet  on  the 
slide. 

5.  Make  a  drawing  of  each  stage  of  development  until 
growth  ceases.     Why  does  the  cell  stop  growing? 

6.  State    in  detail  your  results  with   any  conclusions 
to  be  drawn  a*nd  point  out  the  practical  applications  which 
may  be  made. 

This  method  may  be  used  to  advantage  with  mold 
spores. 

EXERCISE  27.     CHINESE  INK  PREPARATION 

Chinese  ink  may  be  used  to  make  bacteria  more  easily 
visible  microscopically  and  to  aid  in  taking  correct  measure- 
ments. 

Apparatus.  Sterile,  dilute  Chinese  ink;  *  clean  flamed 
glass  slides. 

Cultures.     Pure  cultures  (young  agar  streaks  are  best). 

Method.  1.  Place  one  loopful  of  distilled  water  and 
three  loopfuls  of  sterile  Chinese  ink  in  a  row  on  a  clean 
glass  slide,  about  2  cm.  apart. 

2.  Inoculate    the    loopful    of   water   from   the    original 
culture. 

3.  Distribute    the    organisms    well    with    a    platinum 
needle. 

4.  Then  inoculate  the  adjoining  drop  of  ink  from  the  loop- 
ful of  water,  the  second  drop  of  ink  from  the  first,  etc. 

6.  Stir  each  loopful  of  ink  well  and  then  spread  it  so  as 
to  cover  an  area  about  1  cm.  square. 

*  See  appendix  for  method  of  preparation. 


86  GENERAL  MICROBIOLOGY 

6.  Let  dry.     If  desired  the  specimen  may  be  mounted 
in  Canada  balsam  before  examining. 

7.  Examine  with  either  the  1/7   or  the  oil  immersion 
objective. 

8.  Write  the  name  of  the  organism,  the  date,  and  your 
name  on  the  glass  with  India  ink. 

By  the  use  of  the  Chinese  ink  preparation,  it  is  possible 
to  examine  any  organism  unstained.  Organisms  so  treated 
neither  shrink  nor  in  any  way  change  their  form,  making 


FIG.  31. — Chinese  Ink  Preparation.     (Orig.  Northrup.) 

accurate  measurement  possible.  Stains  often  cause  organ- 
isms to  appear  swollen  or  shrunken. 

The  motility  of  bacteria  may  be  more  easily  demon- 
strated by  adding  a  very  slight  amount  of  this  ink  to  a 
hanging  drop  of  the  organism  being  studied. 

Caution.  Chinese  ink  is  very  expensive.  When  making 
preparations,  use  every  precaution  to  keep  your  supply 
sterile,  as  contaminating  organisms  may  be  confused  with 
the  culture  under  study.  A  control  preparation  to  which 
no  microorganisms  have  been  added  will  serve  to  detect 
their  presence, 


THE  STAINING  OF  MICROORGANISMS  87 


EXERCISE  28.     THE  STAINING  OF  MICROORGANISMS 

Microorganisms  are  devoid  of  color  as  a  rule  and  are 
stained  for  the  purpose  of  observing  their  morphology  to 
better  advantage  than  in  a  hanging  drop.  Staining  also 
often  serves  to  bring  out  certain  morphological  character- 
istics which  are  otherwise  not  evident,  such  as  the  presence 
of  metachromatic  granules  or  a  peculiar  arrangement  of 
the  protoplasm,  resulting  in  what  are  known  as  "  beaded 
forms." 

The  stains  best  suited  to  bacteria  are  the  basic  anilin 
dyes  which  are  derived  from  the  coal-tar  product  anilin 
(CeHsNH^).  Many  of  them  have  the  constitution  of  salts. 

Such  compounds  are  divided  into  two  groups,  according 
as  the  staining  action  depends  on  the  basic  or  the  acid 
portion  of  the  molecule.  Fuchsin,  gentian  violet  and  methy- 
len  blue  are  basic  dyes,  while  eosin,  picric  acid  and  acid 
fuchsin  are  acid  dyes. 

These  groups  have  affinities  for  different  parts  of  the  liv- 
ing cells.  The  basic  stains  have  a  special  affinity  for  the 
nuclei  of  tissues  and  for  bacteria,  the  acid  for  the  proto- 
plasm and  not  for  bacteria.  The  violet  and  the  red  anilin 
dyes  In  order,  are  the  most  intense  in  action,  easily  over- 
staining  the  specimen.  It  is  difficult  to  overstain  with 
methylen  blue.  For  this  reason  this  stain  is  to  be  pre- 
ferred where  the  bacteria  occur  in  thick  or  viscid  substances, 
like  pus,  mucus  or  milk.  In  the  presence  of  alkali,  how- 
ever, the  stain  acts  more  energetically. 

Stock  solutions  of  the  ordinary  dyes  are  commonly 
used.  These  are  prepared  by  making  a  saturated  solution 
of  the  dye  in  absolute  alcohol;  this  is  diluted  with  water 
as  needed. 

Saturated  alcoholic  solutions  of  dyes  will  stain  bacteria 
with  difficulty.  The  best  results  are  obtained  with  the 
diluted  stain,  spoken  of  here  as  an  "  aqueous-alcoholic  " 
stain. 


88  .  GENERAL  MICROBIOLOGY 

Apparatus.  Clean  cover-glasses;  clean  slides;  cover- 
glass  forceps;  platinum  loop  and  needle;  Bunsen  burner; 
small  pieces  of  filter  paper;  distilled  water;  aqueous-alco- 
holic solution  of  fuchsin,  methylen  blue,  etc.;  Canada 
balsam;  microscope. 

Note.     See  appendix  for  formulae  of  stains. 

Method.  1.  Flame  a  clean  cover-glass,  holding  it  by 
one  corner  with  cover-glass  forceps. 

2.  Place  one  loopful  of  distilled  water  in  its  center. 

3.  Touch  the  growth  on  slant  agar  lightly  with  a  ster- 
ilized platinum  needle  and  transfer  a  very  little  of  the  mate- 


FIG.  32. — For  use   in  mounting  permanent  cover-glass  preparations. 

(Orig.) 

rial  to  the  drop,  and  only  sufficient  to  make  it  very  slightly 
cloudy.     - 

4.  Flame  the  needle  and  allow  it  to  cool. 

5.  Spread  the  drop  over  the  entire  cover-glass  with  one 
or  two  strokes  of  a  straight  needle.     In  the  case  of  path- 
ogenic microorganisms  use  a  flat  loop  2  mm.  in  diameter, 
and  limit  the  spreading  to  the  inner  three-fourths  of  the 
cover-glass. 

6.  Allow  to  dry  in  air. 

7.  Fix  the  preparation  on  the  cover-glass  by  passing 
the  cover-glass,  specimen  side  up,  three  times  through  the 
flame   of  a   Bunsen   burner.     The   speed   is  measured  by 
moving  the  cover-glass  and  forceps  in  a  circle  of  1  ft.  diam- 
eter in  one  second. 


THE   STAINING  OF  MICROORGANISMS  89 

8.  Flood    the   entire    specimen-side   of   the    cover-glass 
with  stain,  using  a  pipette. 

9.  Allow  the  stain  to  act  a  short  time. 

Note.  The  time  required  for  staining  varies  so  much  with  the 
different  stains,  different  organisms  and  their  physiological  conditions, 
that  no  exact  time  can  be  given.  In  general,  a  good  specimen  is 
obtained  by  staining  one-half  to  one  minute  with  fuchsin  or  gentian 
violet,  or  one  to  five  minutes  with  methylen  blue. 

10.  Wash  the  specimen  in  running  water. 

11.  Mount    the    cover-glass    in    water,  specimen   side 
down,  on  a  clean  slide. 

12.  Dry  the  upper  surface  of  the  cover-glass  and  take 
up  any  excess  of  water  by  means  of  filter  paper. 

13.  Examine   the   slide   under    the    microscope,    using 
objective  No.  7  and  ocular  No.  1. 

14.  If   satisfactory,    remove    the    cover-glass    carefully 
from  the  slide,  floating  it  off  if  necessary. 

15.  Allow  it  to  dry  in  the  air,  specimen  side  up. 

16.  Place  a  clean  slide  exactly  on  the  figure  (Fig.  32). 

17.  Let  a  small  drop  of  Canada  balsam  fall  in  the  center 
of  the  slide,  marked  by  the  circle. 

Note.  The  consistency  of  the  Canada  balsam  should  be  like  thin 
cream.  The  diameter  of  the  glass  rod  should  not  be  more  than  4  mm. 

18.  Place  the  cover-glass,  specimen  side  down,  on  this 
drop. 

19.  Allow  the  balsam  to  spread  over  the  entire  under 
surface  of  the  cover-glass  (without  pressing  it  down  on  the 
slide)  and  keep  the  cover-glass  straight,  coinciding  with  the 
lines  of  the  figure. 

20.  Label,  stating  in  order,  the  name  of  the  organism, 
the  age  and  kind  of  culture,  the  stain  used,  the  date,  your 
own  name  and  the  purpose  of  the  stain  if  otherwise  than 
ordinary,  e.g.,  spore  stain. 

21.  Allow  the  slide  to  stand  in  a  horizontal  position  for 
a  few  days  until  the  balsam  becomes  hard. 


90 


GENERAL  MICROBIOLOGY 


EXERCISE    29.     ANJESZKY'S    METHOD    OF    STAINING 

SPORES 

Spores  are  not  stained  by  the  ordinary  staining  methods, 
as  their  physical  nature  differs  from  that  of  the  vegetative 
rods  within  which  they  are  formed.  By  proper  treatment 
with  strong  anilin  dyes,  however,  it  is  possible  to  force  the 
stain  into  the  spore.  Once  within  the  spore  it  is  as  dif- 
ficult to  remove  the  dye  as  it  was  to  cause  it  to  enter. 


FIG.  33. — Contrast  Spore  Stain,  Carbol-fuchsin  and  Methylen  Blue, 
X1500.     (Orig.  Northrup.) 

By  a  careful  decolorization  with  a  weak  acid,  it  is  pos- 
sible to  remove  the  stain  from  everything  on  the  cover- 
glass  except  from  the  spores.  Then,  on  application  of  a 
dye  of  a  contrasting  color,  the  specimen  will  show,  e.g.,  a 
bright  red  spore  within  a  blue  bacterium. 

The  fundamental  principles  of  this  method  are  also 
used  for  staining  "  acid-fast  "  organisms,  ^  Bad.  tuber- 
culosis. 

Apparatus.  Clean  cover-glasses;  clean  slides;  cover- 
glass  forceps;  platinum  loop  and  needle;  Bunsen  burner; 


ANJESKY'S  METHOD  OF  STAINING  SPORES        91 

carbol-f uchsin ;  methylen  blue,  aqueous^alcoholic ;  hydro- 
chloric acid,  0.5%;  sulphuric  acid,  5%;  Canada  balsam; 
microscope. 

Culture.  Culture  of  an  organism  just  beginning  to 
show  spore  formation. 

Method.  1.  Prepare  a  cover-glass  film  of  the  spore- 
containing  organism  and  allow  it  to  dry. 

2.  While  it  is  drying,  warm  some  0.5%  HC1  in  the  small 
evaporating   dish   over  a  Bunsen   burner  until  it   steams 
well  and  bubbles  begin  to  form. 

3.  When  the  solution  is  hot  and  the  smear  dry,  drop 
the  cover-glass  upon  the  fluid  and  allow  it  to  act  upon  the 
unfixed  smear  for  three  to  four  minutes. 

4.  Wash  and  dry  the  cover-glass. 

5.  Fix  in  the  flame  for  the  first  time. 

6.  Stain    with    carbol-fuchsin    by    flooding    the    cover- 
glass  with  the  stain,  warming  twice  until  fumes  arise. 

7.  Allow  to  cool,  and  wash  in  water. 

8.  Decolorize  with  5%  H2SO4.     Spores  are  treated  with 
a  mild  decolorizing  agent,  as  they  are  much  less  resistant 
to  acid  than  are  acid-fast  bacteria.    (See  p.  93,  step  7.) 

9.  Wash  in  water. 

10.  Counterstain  for  one  to  two  minutes  with  methylen 
blue. 

11.  Wash,    dry   and   examine   the   specimen   in   water. 
If  satisfactory,  dry  it  and  mount  in  balsam. 

The  whole  procedure  should  not  take  longer  than  eight 
to  ten  minutes. 

REFERENCE 

MCFARLAND:  Textbook  of  Pathogenic  Bacteriology,  p.  188. 


92  GENERAL  MICROBIOLOGY 


EXERCISE   30.     METHOD    OF   STAINING   TUBERCLE 
AND  OTHER  ACID-FAST  BACTERIA 

Acid-fast  bacteria  are  so  termed  from  their  reaction  to 
a  special  staining  process.  This  process  consists  in  staining 
the  specimen  containing,  for  example,  tubercle  bacteria, 
with  hot  carbol-f  uchsin  and  decolorizing  for  a  short  time 
with  acid;  the  acid  takes  the  dye  out  of  all  other  material, 
bacteria  and  blood  or  other  body  cells  that  may  be  present, 
leaving  the  tubercle  bacteria  stained  red.  This  staining 
process  is  essentially  the  same  as  for  spores,  but  the  prin- 
ciple is  different. 

The  property  which  some  bacteria  possess  of  being 
acid-fast  is  attributed  to  the  presence  of  fat  and  wax-like 
substances  in  their  cells.  This  seems  to  be  proved  by  the 
fact  that  when  the  bacterial  cell  substance  of  tubercle 
bacteria  has  been  freed  from  these  fats  and  waxes  by 
extraction  with  absolute  alcohol  and  ether,  this  property 
is  lost. 

Apparatus.  Clean  slides;  clean  cover-glasses;  plati- 
num loop;  copper  staining  dish;  Bunsen  burner;  forceps; 
carbol-f  uchsin;  sulphuric  acid,  20%;  methylen  blue,  aque- 
ous-alcoholic; immersion  oil;  Canada  balsam;  specimen 
to  be  examined. 

Method.  1.  Using  a  sterile  loop,  smear  some  of  the 
specimen  in  the  center  of  one  surface  of  a  clean  slide, 
taking  care  not  to  come  within  0.5  cm.  of  the  edge. 

Note.  This  may  be  applied  to  sputum,  pus,  etc.  In  case  of 
tubercles  or  diseased  organs  or  tissues  these  may  be  cut  open  with  a 
scalpel,  a  portion  incised,  and  grasping  this  portion  with  the  forceps 
a  smear  made  directly  on  the  slide,  following  the  precautions  above. 
If  pure  cultures  are  to  be  examined,  a  cover-glass  specimen  may  be 
made  in  the  usual  way. 

2.  Dry  the  slide  in  air. 

3.  Fix  in  the  flame. 

4.  Support  the  slide  on  the  copper  staining  dish;    flood 


METHOD  FOR  STAINING  FLAGELLA  93 

the   slide    with    carbol-fuchsin    until    the    stain    "  rounds 
up." 

5.  Heat  the  under  side  of  the  slide  directly,  with  a  flame 
until  the  carbol-fuchsin  steams  (but  not  boils).     Keep  the 
stain  steaming  for  five  minutes. 

6.  Wash  in  water. 

7.  Decolorize   by   dipping   the   slide   preparation   into 
20%  H2SC4  for  an  instant  and  washing  immediately.     This 
process  may  have  to  be  repeated  two  or  three  times.     If 
not  careful,  however,  the  tubercle  bacteria  may  be  decolor- 
ized.    If   this   happens,    their   acid-fast   property   will   be 
destroyed  to  some  extent. 

8.  Counterstain  with  aqueous-alcoholic  methylen  blue. 

9.  Wash  in  water,  dry  and  examine  directly  with  the 
oil  immersion  lens.     The  specimen,  if  a  good  one,  may  then 
be  mounted  in  the  usual  way  without  removing  the  immer- 
sion oil. 

EXERCISE  31.     METHOD  FOR  STAINING  FLAGELLA 

Flagella,  the  exceedingly  delicate  organs  of  locomotion 
of  bacteria,  cannot  be  seen  in  an  unstained  or  in  an  ordinary 
stained  preparation.  Special  staining  methods  must  be 
employed  to  make  them  visible.  They  are  generally  ren- 
dered visible  by  precipitating  some  chemical  on  them; 
this  generally  increases  their  width  considerably. 

The  staining  of  the  flagella  of  bacteria  is  the  most 
difficult  of  all  bacteriological  procedures  and  it  generally 
requires  considerable  practice  to  insure  good  results. 

There  are  many  methods  for  staining  flagella.  This 
one,  however,  has  met  with  considerable  success  with  stu- 
dents. Failure  to  make  a  good  flagella  stain  with  any 
method  is  no  sign  that  the  student  is  not  a  good  work- 
man, nor  is  success  the  sign  of  a  good  bacteriologist. 

Apparatus.  Clean  glass  slides;  absolutely  clean  cover- 
glasses;  small  platinum  loop;  several  cover-glass  forceps; 


94  GENERAL  MICROBIOLOGY 

distilled  water;   mordant  for  flagella  staining;    anilin-water 
fuchsin  or  gentian  violet. 

Culture.   Agar  slant  culture,  twelve  to  eighteen  hours  old. 

Note.  The  best  results  are  obtained  if  successive  generations  of 
this  organism  have  been  transplanted  every  eighteen  to  twenty-four 
hours  for  several  generations. 

Method.  1.  Place  three  drops  of  distilled  water  on  a 
clean  glass  slide. 

2.  Transfer  a  small  amount  of  bacterial  material  from 


-  FIG.  34. — Flagella  Stain.     B.  typhosus,   X1500. 

the  moist  portion  of  the  agar  slant  culture  to  the  first  drop 
by  means  of  a  small  platinum  loop,  using  only  enough  of 
the  material  to  make  this  drop  very  slightly  cloudy. 

3.  Flame  the  needle  and  transfer  a  small  portion  from 
the  first  drop  to  the  second. 

4.  Proceed  in  like  manner  in  preparing  the  third  dilu- 
tion. 

5.  Place  a  number  of  absolutely  clean  cover-glasses  in 
cover-glass  forceps. 

6.  By  means  of  a  platinum  needle  bent  at  right  angles 
near  the  end,  make  smears  on  the  cover-glasses  from  the 


GRAM'S  METHOD  OF  STAINING  95 

second  and  third  drops  on  the  slide  by  drawing  the  bent 
needle  once,  lightly  across  the  cover-glass.  //  there  is  any 
tendency  of  the  smear  to  roll  or  "  gather  in  drops,"  the  cover- 
glass  should  be  discarded  and  a  clean  one  substituted.  This 
is  imperative. 

7.  Allow  the  preparation  to  dry  for  about  five  min- 
utes. 

8.  Filter  on  each  of  the  fixed  smears  enough  of  the  mor- 
dant to  cover  the  cover-glass. 

9.  Allow  to  stand  about  five  minutes  at  room  tempera- 
ture. 

10.  Wash    off    the    mordant    in    a    small    stream    of 
water. 

11.  Draw  off  the  excess  water  from  the  edge  of  the  cover- 
glass  by  means  of  filter  paper. 

12.  Stain  with  anilin-water  fuchsin  or  anilin-water  gen- 
tian violet  for  about  five  minutes,  either  cold  or  by  warm- 
ing somewhat  over  a  low  flame. 

13.  Wash  off  the  excess  stain  with  clean  water. 

14.  Mount  on  a  slide  in  water. 

15.  Absorb  the  excess  water  with  filter  paper. 

16.  Examine   under   the   microscope.     If  the   prepara- 
tion has  been  successful,  it  may  be  dried  and  mounted 
in  balsam, 

EXERCISE  32.     GRAM'S  METHOD  OF  STAINING 

Certain  organisms,  when  stained  with  anilin-water 
gentian  violet  and  afterwards  treated  with  a  solution  of 
iodin  and  washed  in  alcohol  or  anilin  oil,  give  up  the  stain; 
others  retain  the  color  when  subjected  to  this  process. 
These  latter  organisms  are  said  to  be  Gram-positive,  those 
losing  the  stain  are  Gram-negative. 

This  phenomenon  is  interpreted  by  Benians  to  be  due 
to  the  possession  of  a  definite  cell-envelope  which  by  the 
action  of  iogiin  is  rendered  impermeable  to  alcohol.  His 


96  GENERAL  MICROBIOLOGY 

experiments  show  that  so  long  as  the  Gram-stained  cell 
is  intact,  the  solvent  is  unable  to  remove  the  stain,  but 
that  as  soon  as  the  cell  is  crushed  and  injured,  the  stain 
is,  in  great  part,  dissolved  out.  The  amorphous  debris 
obtained  from  broken-up  Gram-positive  bacteria  does  not 
retain  Gram's  stain. 

Apparatus.  Clean  slides;  clean  cover-glasses;  plati- 
num loop  and  needle;  cover-glass  forceps;  distilled  water; 
anilin- water  gentian  violet;  Lugol's  iodin  solution;  aceton- 
alcohol. 

Culture.     Agar  slant  cultures  preferably. 

Method.  1.  Prepare  a  cover-slip  film  and  fix  in  the 
usual  way. 

2.  Stain   in   anilin-water  gentian  violet   three   to   five 
minutes. 

3.  Wash  in  water. 

4.  Treat  with  Lugol's  iodin  solution  until  the  film  is 
black  or  dark  brown. 

5.  Wash  in  water. 

6.  Dry  in  air. 

7.  Wash  in  aceton-alcohol  until  no    more  color  is  dis- 
charged. 

8.  Wash  in  water.   (Counterstain  at  this  point  if  desired.) 

9.  Dry  in  air. 

10.  Mount  in  Canada  balsam. 

Note.  The  Gram-Weigert  method  is  more  applicable  in  case  of 
sections  of  tissues.  The  directions  from  1-6  are  the  same.  The  speci- 
men is  washed  in  anilin  oil  1  part,  xylol  2  parts,  instead  of  alcohol, 
washed  further  in  xylol  and  mounted  at  once  in  Canada  balsam.  Bad. 
bidgaricum  in  milk  is  very  beautifully  demonstrated  by  this  modified 
method. 

A  few  of  the  more  common  Gram-positive  and  -negative 
organisms  are  appended.  This  is  not  as  important  a  diag- 
nostic method  as  has  been  formerly  supposed,  because 
the  reaction  occurring  often  depends  upon  the  age  of  the 
culture,  the  medium  on  which  it  is  grown,  etc. 


METHOD  FOR  STAINING  CAPSULES  97 

GRAM-POSITIVE  ORGANISMS.      GRAM-NEGATIVE  ORGANISMS. 

Staph.  pyogenes  aureus  and  albus  Bact.  mallei 

Strept.  pyogenes  Bact.  aerogenes 

Bact.  anthracis  B.  typhosus 

Bact.  tuberculosis  B.  coli  communis 

B.  alvei  B.  cholera?  suis 

B.  tetani  M.  gonorrhea? 

Bact.  acidi  lactici  Msp.  deneke 

Bact.  bulgaricum  Msp.  finkler-prior 

B.  megaterium  Spirocheta  obermeieri 

B.  subtilis  Proteus  tulgaris 

B.  myeoides  Ps.  medicaginis 

B.  mesentericus  vulgatus  B.  amylovorus 

M.  tetragenus  Ps.  campestris 

Streptothrix  actinomyces  B.  phytophthorus 

Sacch.  cerevisice  and  other  yeasts  B.  caratovorus 

Molds 

REFERENCE 

BENIANS,  T.  H.  C.:  "Observations  on  the  Gram-positive  and  Acid- 
fast  Properties  of  Bacteria."  Jour,  of  Path,  and  Bact.,  Vol. 
XVII,  pp.  199-211  (1912). 

EXERCISE  33.     METHOD  FOR  STAINING  CAPSULES 

Some  bacteria  possess  a  gelatinous  envelope  or  "  cap- 
sule "  which  in  some  species  surrounds  each  individual 
organism,  and  in  others,  groups  of  organisms.  The  pres- 
ence of  this  capsule  may  be  demonstrated  by  various 
special  staining  methods.  The  capsule  takes  the  stain 
much  less  quickly  than  does  the  organism,  leaving  a  light- 
colored  halo  about  it.  The  presence  of  a  capsule  does  not 
always  indicate  that  the  organism  forming  it  is  a  slime- 
forming  organism,  nor  does  the  fact  that  an  organism  is  a 
slime-former  preclude  the  possession  of  a  capsule. 

Apparatus.  Clean  cover-glasses;  clean  slides;  platinum 
loop;  cover-glass  forceps;  filter  paper,  pieces;  glacial 
acetic  acid;  gentian  violet,  aqueous-alcoholic. 


98  GENERAL  MICROBIOLOGY 

Cultures.     Cultures  in  milk,  serum,  etc.,  media. 

Method.  1.  Prepare  the  cover-glass  specimen  directly 
from  the  medium  without  the  use  of  water.  Spread  and  fix 
in  the  usual  manner. 

2.  Flood  the  specimen  side  of  cover-glass  with  glacial 
acetic  acid. 

3.  Drain    immediately   without    washing.     A    piece    of 
filter  paper  may  be  touched  to  the  edge  of  glass  to  take 
up  surplus  water  and  facilitate  drainage. 

4.  Stain    with    aqueous-alcoholic    gentian   violet   for   a 
few  seconds. 

5.  Examine  under  the  microscope. 

6.  Wash,  dry  and  mount, 

EXERCISE    34.     METHOD    OF    MAKING    IMPRESSION 
PREPARATIONS 

Impression  preparations  (Klatschpraparat)  are  pre- 
pared from  isolated  colonies  of  bacteria  in  order  that  their 
characteristic  formation  may  be  examined  by  higher  powers 
than  can  be  used  with  the  living  cultivation  in  situ.  They 
are  prepared  from  plate  cultivations.  Young  colonies  of 
Bad.  anthracis  produce  beautiful  impression  preparations. 

Apparatus.  Clean  cover-glasses;  clean  slides;  Novy 
cover-glass  forceps;  dissecting  needle;  stain. 

Culture.  Agar  plate  culture  containing  well-isolated 
colonies  of  organism  to  be  studied. 

Method.  1.  Taking  a  clean  cover-glass  in  the  Novy 
forceps,  open  the  plate  and  rest  one  edge  of  the  cover- 
glass  on  the  surface  of  the  medium  a  little  to  one  side  of 
the  selected  colony. 

2.  Lower  it  carefully  over  the  colony  until  horizontal. 
Avoid  any  lateral  movement  or  the  inclusion  of  air  bubbles. 

3.  Press  gently  on  the  center  of  the  upper  surface  of 
the  cover-glass  with  the  points  of  the  forceps  to  insure 
perfect  contact  with  the  colony. 


STAINING  THE   NUCLEI  OF  YEAST  CELLS          99 

4.  Steady  one  edge  of  the  cover-glass  with  the  forceps 
and  pass  the  point  of  the  dissecting  needle  just  under  the 
opposite  edge  and  raise  carefully;  the  colony  will  be  adher- 
ent to  it. 

When  nearly  vertical,  grasp  the  cover-glass  with  the 
forceps  and  remove  it  from  the  plate.  Re-cover  the 
plate. 

5.  Place  the  cover-glass  specimen  side  up  on  desk  and 
cover  with  half  a  Petri  dish  until  dry. 

6.  Fix  in  the  flame. 

7.  Stain  and  mount  as  with  ordinary  cover-glass  speci- 
men, being  careful  to  perform  all  washing  operations  with 
extreme  gentleness. 

EXERCISE  35.     METHOD  OF  STAINING  THE  NUCLEI 
OF  YEAST  CELLS 

The  nuclei  of  yeast  cells  are  not  visible  in  unstained  or 
in  ordinary  stained  specimens.  A  special  method  of  pro- 
cedure must  be  used. 

Apparatus.  Clean  cover-glasses;  clean  slides;  forceps; 
ferric  ammonium  sulphate,  3%  aqueous  solution;  Ehrlich's 
hematoxylin  solution;  two  staining  dishes  for  slides. 

Culture.     Culture  of  Saccharomyces  or  Torula. 

Method.  1.  Prepare  and  fix  the  film  upon  the  slide  in 
the  usual  way. 

2.  Soak  in  3%  ferric  ammonium  sulphate  "for  two  hours. 

3.  Wash  thoroughly  in  water. 

4.  Stain  in  hematoxylin  solution  for  thirty  minutes. 

5.  Wash  in  water. 

6.  Differentiate  in  ferric  ammonium  sulphate  solution 
for  one  and  a  half  to  two  minutes,  examining  wet   under 
the  microscope  during  the  process. 

7.  Wash,  dry  and  mount. 


100  GENERAL  MICROBIOLOGY 


GENERAL     CHARACTERISTICS     OF     MOLD     GROWTH 
AND  HINTS  FOR  STUDY 

A  brief  description  of  the  molds  to  be  studied  in  the 
laboratory  is  here  given.  The  references  cited  will  give 
more  in  detail  of  their  structure,  importance  and  occur- 
rence. 

In  these  descriptions,  there  have  been  noted  the  parts 
of  the  structure  of  each  mold  that  are  to  be  found  micro- 
scopically and  drawn,  also  the  quickest  method  of  obtain- 
ing the  best  results.  All  microscopic  drawings  and  meas- 
urements can  be  secured  from  the  adhesion  culture  or  the 
moist-chamber  culture. 

Rhizopus  nigricans — Black  mold 
(Mucor  stolonifer) 

The  mycelium  in  the  advanced  stage  consists  of  rhizoids 
(rootlets),  bearing  clusters  of  sporangiophores,  joined  by 
long  hyphse  (the  stolons)  to  the  mycelium  proper.  The 
hyphse  are  non-septate. 

The  fruiting  bodies  consist  of  typical  sporangia  (spore 
cases  containing  spores)  borne  on  the  enlarged  end  (col- 
umella)  of  the  sporangiophore.  Spores  are  liberated  by  the 
bursting  of  the  sporangium. 

The  columella  can  be  observed  in  fruiting  bodies  of  a 
light  brown  color;  white  sporangia  are  too  young  and  black 
too  old  to  show  this  structure.  If  no  fruiting  bodies  grow 
in  the  adhesion  culture,  they  may  be  studied  directly  from 
a  plate  culture  by  preparing  a  glycerin  slide.  Take  care 
not  to  burst  the  sporangium  when  transferring  it  to  the 
slide. 

Aspergillus  niger — Black  mold 

The  mycelium  of  this  mold  consists  of  septate  hyphse 
with  frequent  dichotomous  branching. 


CHARACTERISTICS  OF  MOLD  GROWTH  101 

The  fruiting  body  (asexual)  consists  of  an  erefet-  condio- 
phore  usually  ending  more  or  less  abruptly  in  a  dilation  or 
head  which  bears  closely  packed  sterigmata  each  of  which 
in  turn  bears  a  single  chain  of  conidia,  the  newly  formed 
conidium  being  pushed  away  by  the  formation  of  a  new 
spore;  thus  the  conidium  at  the  end  of  the  chain  is  the 
oldest.  The  conidia  of  this  mold  are  black. 

Penicillium  italicum — Blue-green  mold 

The  mycelium  consists  of  septate  hyphae,  having  fre- 
quent dichotomous  branching. 

The  conidial  fructification  resembles  a  brush,  the  conidia 
(spores)  being  borne  on  the  end  of  conidiiferous  cells  (ster- 
igmata) ;  in  this  genus  before  the  conidia  appear,  there  is 
generally  a  primary  and  even  a  secondary  branching  of  the 
condiophore  in  some  species  before  the  conidiiferous  cells 
are  formed.  The  species  of  Penicillium  have  more  of  a 
brush-like  appearance  than  the  species  of  Aspergillus.  The 
spores  of  P.  italicum  are  blue-green. 

Oospora  (Oidium)  lactis — White  mold 

The  mycelium  consists  of  septate  hyphse,  having  di- 
chotomous branching;  the  hyphse  are  almost  entirely  sub- 
merged in  the  nutrient  substrate. 

This  differs  from  the  other  molds  in  that  it  does  not  have 
typical  fruiting  bodies.  It  reproduces  by  means  of  conidia, 
which  are  formed  by  a  simple  division  of  the  hyphse. 
The  conidia  are  colorless. 

REFERENCES 

MARSHALL:  Microbiology,  pp.  12-27. 

LAFAR:  Technical  Mycology,  Vol.  II,  Part  I,  pp.  5,  15,  71-77;  Part  II, 

pp.  300-346,  451-455. 
KLOCKER:  Fermentation  Studies,  pp,  184,  185,  274-282,  303,  304. 


PLATE  I* 


Germination  of  Rhizopus  Spore,  Mycelium,  Rhizoids  and  Development 
of  Sporangiophores  and  Sporangium. 


Ripe  Sporangium. 


PLATE  II* 


103 


Sporangiophore    with    Columella    Attached    and"  Ripe    Sporangium 
Showing  Spores  Within. 


Various  Stages  in  the  Formation  and  Germination  of  a  Zygospore. 

*  Plates   I-VI   photographed  from   models   manufactured  by  R. 
Brendel,  Berlin — Grunewald,  Bismarck — Allee  37,  Germany. 


PLATE  III 


MICROSCOPICAL  EXAMINATION  OF  MOLDS       105 

EXERCISE     36.     MICROSCOPICAL    EXAMINATION     OF 

MOLDS 

Apparatus.  Clean  cover-glasses;  clean  slides;  hand 
lens  or  compound  microscope;  platinum  needle  and  loop; 
dissecting  needle;  glycerin,  10%. 

Cultures.     Plate  culture  of  mold. 

Method.  The  gross  structure  of  a  mold  colony  upon 
a  plate  may  be  examined  with  a  hand  lens  or  by  placing 
the  inverted  Petri  dish  culture  on  the  stage  of  the  compound 
microscope  and  examining  with  objective  No.  3  and  ocular 
No.  1.  The  structure  may  be  examined  in  detail  as  follows: 

1.  Select  a  young  colony  which  shows  colored  fruiting 
bodies,  if  such  are  produced  by  the  organism  to  be  studied. 
(Growth  from  natural  or  artificial  media  may  be  treated 
in  the  same  general  way.) 

2.  Using   a   sterile   platinum   needle,    transfer   a   small 
portion  of  the  mycelium  and  fruiting  bodies  to  a  drop  of 
glycerin    on   a   plain   glass  slide.     If  the  mold  growth   is 
closely  confined  to  the  surface  of  the  media  (as  with  Peni- 
cillium  or  Aspergillus) ,  it  is  often  desirable  to  cut  out  a 
small  piece  of  the  medium  bearing  the  mold  and  lift  to  the 
slide  by  means  of  a  sterile  platinum  loop. 

3.  Tease  out  very  gently,  using  dissecting  needles  or 
common  pins.     The  mold  structure  is  extremely  delicate, 
so  this  operation  must  be  performed  with  the  utmost  care. 

4.  Place  a  cover-glass  over  the  preparation. 

5.  Examine  with  the  microscope,  using  objective  No.  3 
and  ocular  No.  1.     When  a  portion  of  mycelium  bearing 
fruiting  bodies  is  found,   examine  with  objective  No.   7. 
Draw  the  young  and  the  old  fruiting  organs. 


DESCRIPTION  OF  PLATE  III 
Aspergillus,    Showing    Septate    Mycelium,    Conidiophore    with 

Conidia,  also  Formation  of  As:ogonium. 

Penicillium,  Germination  of  Spore,  Formation  of  Mycelium, 

Septate  Conidiophores  with  Conidia,  Ripe  Ascospore, 


PLATE  IV 


THE  STUDY  OF  MOLDS  107 


EXERCISE  37.     THE  STUDY  OF  MOLDS 

Apparatus.  Ten  sterile  Petri  dishes;  four  tubes  of 
sterile  slanted  agar;  ten  tubes  of  sterile  agar,  for  plates; 
four  tubes  of  sterile  cider  or  wort;  four  tubes  of  sterile 
gelatin;  clean  glass  rings,  slides  and  cover-glasses;  hand 
lens;  compound  microscope;  centimeter  scale. 

Cultures.  Pure  or  mixed  cultures  of  the  following  four 
molds:  Rhizopus  nigricans;  Aspergillus  niger;  Penicillium 
italicum;  Oospora  lactis.  Mixed  (or  impure)  cultures  of 
two  molds  growing  in  their  natural  habitat  will  be  found 
on  each  table. 

Method.  1.  Plate  out  each  mixed  culture*  making  three 
straight  needle  dilution  plates  for  each.  Use  agar  as  a 
medium.  Place  the  plates  in  the  constant-temperature  room 
in  the  place  assigned.  Note  the  temperature. 

2.  When  the  plates  are  twenty-four  hours  old,  mark 
and  draw  a  well-isolated  typical  colony  of  the  mold  from  the 
most   thinly   populated   plate.     Measure   and   record   the 
diameter  of  the  colony  in  millimeters. 

3.  When  fruiting  bodies  begin  to  show,  isolate  a  pure 
culture  of  each  mold  in  cider  (see  Exercise  16). 

4.  (a)  As  soon  as  growth  begins  to  show  in  the  tubes  of 
cider    (about    twenty-four    to    thirty-six    hours)    make    a 
macroscopic  drawing  of  each.     State  the  age  of  the  culture. 

(b)  When  mycelium  is  well  developed  and  fruiting 
bodies  appear  (as  noted  on  plates)  make  a  second  drawing. 

*  Two  mixed  and  two  pure  cultures  are  furnished  for  study.  These 
cultures  owe  their  color  to  the  presence  of  fruiting  bodies  or  spores. 
Always  endeavor  to  obtain  spores  when  making  inoculations  from 
molds. 

DESCRIPTION  OF  PLATE  IV 

Sexual  Reproduction  of  Penicillium,  Ascus  Formation. 
Formation  of  Chains  of  Asci  in  Process  of  Ripening,  Some  Con- 
taining Ascospores,  Section  of  a  Ripe  Ascus. 
Saccharomyces,    Budding,    Colony   Formation,    Produc- 
tion of  Ascus  and  Germination  of  Ascospores. 


108 


GENERAL  MICROBIOLOGY 


FIG.  35. — Oospora  (Oidium)  lactis  (after  Hansen)  see  p.  104,  Jorgensen. 

1.  Hyphae  with  forked  partitions;  2,  two  ends  of  hyphse — one  with 
forked  partition,  the  other  with  commencement  of  development 
of  a  spherical  link;  3-7,  germinating  conidia;  6-6'",  germination  of 
a  conidium,  sown  in  hopped  beer-wort  in  Ranvier's  chamber, 
and  represented  at  several  stages;  at  each  end  germ  tubes  have 
developed;  after  nine  hours  (6'")  these  have  formed  transverse 
septa  and  the  first  indications  of  branchings;  11-14,  abnormal 
forms;  15,  16,  hyphsn  with  interstitial  cells,  filled  with  plasma; 
17,  chain  of  germinating  conidia;  18,  conidia  which  have  lain 
for  some  time  in  a  sugar-solution;  the  contents  show  globules 
of  oil;  19,  old  conidia. 


THE  STUD.Y  OF  MOLDS  109 

State  the  age  of  the  culture.     These  cider  tubes  are  then  of 
the  proper  age  from  which  to  make  inoculations. 

5.  Pure   cultures   of  the   two  remaining  molds  will   be 
found  in  tumblers  marked  "  Laboratory  Cultures." 

Always  leave  such  cultures  in  the  place  assigned,  after 
using. 

Make  cultures  of  each  of  the  four  molds  as  follows: 

(a)  Agar  slant  (for  method  see  Exercise  15). 

(b)  Agar  plate  (giant  colony) .     Use  Roux  culture  flask  for 
Rhizopus  nigricans  (see  pp.  2,  62). 

(c)  Cider  or  wort  (test-tube  culture).*     For  method  of 
inoculation  of  c  and  d  see  pp.  60,  61. 

(d)  Gelatin  stab  (test-tube  culture).  Keep  all  gelatin  test- 
tube  cultures  in  cold-water  bath  or  in  a  cool  place  (15°  to  20°  C). 

(e)  Adhesion  or  moist-chamber  culture.    (See  pp.  78-81). 
Prepare  these  cultures  from  the  freshly  inoculated  cider  tubes. 

6.  Make  drawing  of  spores  of  each  mold  from  adhesion 
or  moist-chamber  culture  as  soon  as  preparation  is  made. 
Measure  the  spores  and  record  the  limits  of  size. 

7.  Draw  the  twenty-four  hour  cultures  each  of  a,  b,  c,  d, 
and  e  and  label  in  detail.     Measure  the  spores  which  have 
germinated  in  e,  and  record  the  diameter  and  length  of  the 
mycelium. 

8.  Make  drawings   of  branched  mycelium  and  several 
stages  of  development  of  fruiting  bodies  from  a  glycerin 
mount.     This  mount  is  most  easily  prepared  from  an  agar 
plate  colony. 

9.  Make  drawings  of  all  cultures  as  soon  as  a  marked 
development  is  seen  over  that  of  the  preceding  drawing. 
Three  drawings  of  each  culture  should  be  sufficient. 

10.  Measure    giant    colony    of  each    mold  every   day 
and  record  the  measurements.     What  is  noted  of  the  com- 
parative rate  of  growth? 

All  drawings  must  be  made  directly  on  charts  or  in  note- 

*  Cider  cultures  have  already  been  made  of  the  two  molds  isolated 
from  mixed  culture. 


110 


GENERAL  MICROBIOLOGY 


books  as  assigned.  Describe  the  drawings  at  the  time  they 
are  made.  Descriptions  are  to  be  recorded  in  ink;  use  a  6H 
pencil  for  drawings. 

This  outline  or  some  modification  of  it  may  be  employed 
for  the  various  species  of  molds. 

MOLDS 


Name  of  student 

Desk  No. 

Name  of  organism 

Isolated  from 

Method  of  isolation 

Occurrence 

Importance 

Spore 

Stages  of  germination 


Drawn  from preparation 


Mycelium 


Drawn  from 


.  preparation 


Method  of  reproduction 


Drawn  from preparation 


Total  organism 


Drawn  from preparation 


Note:  Mold,  yeast  and  bacteria  charts  (pp.  110-111,  122-123,  135- 
138)  may  be  procured  from  the  College  Printery,  East  Lansing,  Mich. 


THE  STUDY  OF  MOLDS 


111 


Cider  or  wort 
culture 
Titre    

Iday 

days 

days 

Incubated  at 
C. 

^J 

0 

[^ 

Nutrient  gelatin 
stab 
Titre 

1  day 

days 

.....days 

Incubated  at 

c. 

^J 

^J 

^J 

A  gar  streak 
Titre 

1  day 

r\ 

.days 

r\ 

days 

r\ 

Incubated  at 

°c. 

^ 

^ 

^ 

Age  of  colony 

Size  of  colony 

Surface  elevation 

Gelatin  or  agar 
colony 
Titre    

Incubated  at 

112  GENERAL  MICROBIOLOGY 


EXERCISE  38.  TO  DETERMINE  THE  ACIDITY  CHANGES 
PRODUCED  BY  MOLDS  IN  CIDER  (OR  OTHER 
LIQUID  MEDIA  HAVING  A  LOW  ACIDITY  AND 
LOW  SUGAR  CONTENT) 

Apparatus.  Four  100  c.c.  Erlenmeyer  flasks;  200  c.c. 
of  cider;  six  1-c.c.  pipettes  for  titration  (sterile);  normal 
NaOH;  N/20  NaOH. 

Cultures.     Pure  cultures  of  four  molds. 

Method.  1.  Determine  the  titre  (reaction)  of  the  cider 
and  neutralize  with  normal  sodium  hydrate. 

2.  Place  50  c.c.  in  each  of  the  Erlenmeyer  flasks  and 
sterilize  by  the  Tyndall  method. 

3.  Inoculate  each,  using  a  different  mold  for  each  flask. 

4.  As  soon  as  the  mold  mycelium  shows  in  the  flask 
(examine  by  looking  through  the  flask  toward  the  light), 
titrate. 

Note.  One  cubic  centimeter  of  neutral  cider  is  diluted  to  50  c.c. 
with  distilled  water  for  titrating  as  the  larger  quantity,  5  c.c.,  when 
diluted,  is  of  such  a  dark  color  that  it  is  practically  impossible  to  obtain 
a  uniform  or  satisfactory  end  point.  The  burette  reading  must  be 
multiplied  by  5. 

Each  pipette  must  be  used  only  once.  After  using  a  pipette  once, 
clean  and  resterilize  it  for  future  use. 

5.  Titrate  every   three   days   thereafter,   making  eight 
titrations  in  all. 

6.  Tabulate  -your  results.     Plot  a  curve  showing  the  rise 
in  acidity,  making  all  curves  on  one  sheet,  starting  from  the 
same  zero  point,  and  using  different  inks  or  different  kinds 
of   lines   to   represent   the   different   acidity   curves.     Use 
acidity  values  as  ordinates,  days  as  abscissae. 

7.  State    fully  any   conclusions    which   may  be  based 
upon  your  data  and  point  out  the  practical  application 
which  may  be  made. 

REFERENCE 
LAFAR:  Technical  Mycology,  Vol.  II,  Part  II,  pp.  353-361. 


THE  PATHOGENIC  NATURE  OF  MOLDS  113 


EXERCISE  39.     TO  DEMONSTRATE  THE  PATHOGENIC 
NATURE  OF  MOLDS 

Apparatus.  One  deep  culture  dish;  one  perfect  fruit 
the  same  as  that  from  which  the  mold  was  isolated,  or 
any  fruit  which  is  the  natural  habitat  of  the  mold. 

Cultures.     Pure  culture  of  a  mold  isolated,  from  a  fruit. 

Method.  1.  Make  small  circles  on  opposite  sides  of 
the  fruit  with  the  wax  pencil. 

2.  Puncture    the   center   of   one   circle   with   a   sterile 
platinum  needle. 

3.  Then  with  needle  contaminated  with  the  mold  spores 
inoculate  the  circle  on  the  opposite  side  by  puncturing  as 
in  2. 

4.  Place  at  about  25°  C.  and  observe  from  day  to  day 
for  two  weeks. 

5.  How  do  fruits  usually  become   contaminated  with 
molds?    What   preventive   measures   would   you   suggest? 

6.  What    is   a    perfect   fruit    from   the    bacteriological 
standpoint?      From    the    horticultural    standpoint?      May 
these  view-points  differ?     If  so,  how?     What  other  fruits 
would  be  susceptible  theoretically  to  the  mold  you   used? 
Why? 

What  other  types  of  microorganisms  may  be  path- 
ogenic to  fruits? 

7.  State  in  full  the  results  obtained,   with   any   con- 
clusions that  may  be  drawn,  and  point  out  the  practical 
application  which  may  be  made. 

REFERENCES 

MARSHALL:  Microbiology,  p.  513. 

SMITH,  ERWIN  F.:  Bacteria  in  Relation  to  Plant  Diseases,  Vol.  I,  p.  202, 

Plates  29,  30  and  31. 
SMITH,  ERWIN  F.:  Bacteria  in  Relation  to  Plant  Diseases,  Vol.  II, 

Fig.  13,  pp.  60  and  174-181. 


114  GENERAL  MICROBIOLOGY 


YEASTS 

The  so-called  yeasts  are  divided  into  true  yeasts  "  Sac- 
charomycetes "  (wild  and  cultivated),  and  pseudo-yeasts 
or  false  yeasts,  "  Torulce  "  and  "  Mycodermata." 

By  true  yeasts  are  meant  those  which  usually  produce 
alcoholic  fermentation  (Sacch.  membrancefaciens  is  an  ex- 
ception), and  which  are  able  to  form  endospores. 

Pseudo-yeasts  do  not  form  endospores  and  produce 
little  or  no  alcoholic  fermentation. 

Sacch.  cerevisice,  the  yeast  used  in  the  manufacture  of 
beers  and  in  bread-making,  is  a  good  example  of  the  culti- 
vated yeast. 

Sacch.  apiculatus  and  Sacch.  ellipsoideus  are  examples 
of  wild  yeasts  which  are  necessary  in  the  making  of  wines. 
(These  yeasts  are  cultivated  and  pure  cultures  used  to  some 
extent.) 

Torula  rosea  is  an  example  of  the  pseudo-yeast.  These 
look  like  true  yeasts,  reproduce  by  budding,  but  seldom 
produce  alcoholic  fermentation. 

REFERENCES 

MARSHALL:  Microbiology,  pp.  28-36,  420-423,  440,  460. 
KLOCKER:  Fermentation  Studies,  pp.  205,  249,  289,  296. 

EXERCISE  40.  TO  ISOLATE  A  PURE  CULTURE  OF  SAC- 
CHAROMYCES  CEREVISICE  AND  TO  STUDY  THE 
FLORA  OF  A  COMPRESSED  YEAST  CAKE 

Apparatus.  Cover-glasses;  concave  slide;  sterile 
Esmarch  dishes;  potato  knife;  platinum  needles;  Bunsen 
burner;  sterile  pipette;  three  tubes  of  sterile  dextrose 
agar;  iodin  solution;  methylen  blue  (0.0001%  aqueous 
solution) . 

Culture.     Fresh  compressed  yeast  cake. 


ISOLATION  OF  SACCH.   CEREVISLE  115 


A.  Isolation  of  Saccharomyces  Cerevisiae 

Method.  1.  Sterilize  the  potato  knife  in  the  flame  of 
the  Bunsen  burner. 

2.  As  soon  as  cool,  cut  a  piece  off  the  yeast  cake. 

3.  Make  three  dilution  plates  in  dextrose  agar  imme- 
diately   from    this   freshly    cut   surface.     Use   the   straight 
needle  and   transfer  only  a  very  minute  quantity  of  the 
yeast.     Distribute    well    with    the    platinum   needle.     Use 
the  straight  needle  for  making  dilutions  in  all  cases. 

4.  When  the  colony  develops  (three  to  six  days)  examine 
under  objective  No.  3,  ocular  No.  1,  inverting  the  plate 
for  this  purpose. 

The  individual  cells  of  most  yeast  colonies  may  be 
seen  under  objective  No.  3,  while  individual  bacteria  can 
seldom  be  distinguished  in  the  colony  at  this  low  magni- 
fication. 

5.  When  you  have  located  a  yeast  colony  make  a  hang- 
ing drop  from  it  in  water  and  determine  the  shape  of  the 
individual  yeast  cells. 

6.  If  they  have  the   shape  and  size  of  Sacch.  cerevisice 
(see  Marshall,  p.  32),  inoculate  a  tube  of  wort  from  this 
colony. 

7.  Study  this  yeast  according  to  directions  in  Exercise 
42. 

B.  Study  of  Flora  of  Compressed  Yeast  Cake 

Method.  1.  After  preparing  plates,  place  the  yeast 
cake  in  a  sterile  Esmarch  dish. 

2.  Add  1  c.c.  of  boiled  water,  using  a  sterile  pipette. 

3.  From  the  freshly  cut  surface,  prepare  a  hanging  drop 
of  the  yeast  in  water,  adding  a  loopful  of   iodin  solution 
to  it.     Yeast  cells  will  be  unstained,  while  starch  grains 
become  blue. 

4.  Repeat  every  seven  days. 


116  GENERAL  MICROBIOLOGY 

6.  Is  the  cake  made  up  mostly  of  starch  grains  or  yeast 
cells?  What  is  the  purpose  of  the  starch  in  the  yeast 
cake?  Do  the  starch  grains  remain  intact  or  do  they 
disappear?  Explain.  What  kinds  of  starch  are  used? 

6.  Draw  and  measure  the  starch  grains.     A  drawing  of 
the  individual  yeast  cell  may  be  made  from  this  mount. 

7.  Prepare  a  second  hanging  drop  of  yeast  in  water  from 
the  fresh  cake. 

8.  Stain    by    adding    a    loopful    of    0.0001%    aqueous 
methylen  blue.     Dead  yeast  cells  are  stained  blue,  while  the 
living  cells  remain  unstained. 

9.  Count  the  number  of  living  and  dead  cells  in  each  of 
several  fields.     Estimate  the  per  cent  of  living  and   dead 
yeast  cells. 

10.  Repeat  every  seven  days  until  all  the  yeast  cells 
are  dead. 

11.  How  long  does  this  take?    What  factors  influence 
the  death  rate?    Do  other  microorganisms  enter?     If  so, 
what   types?      Why?       From    what    source?       Do    they 
influence  the  value  of  the  yeast  cake?     How? 

12.  Each  time  you  record  the  percentage  of  living  and 
dead  cells,  note  the  macroscopical  appearance  of  the  cake. 
Also  note  ,the  presence  of  new  microorganisms,  consistency 
of  the  cake,  odor,  and  color. 

13.  Record  the  results  of  this  experiment  in  tabulated 
form,   and  state,  any  conclusions  that  may  be  drawn  or 
practical  application  to  be  made. 

REFERENCES 

IAGO,  WM.  and   IAGO,  WM.    C.:    The   Technology  of   Breadmaking. 

(1911),  pp.  235-239. 

CONN:  Yeasts,  Molds  and  Bacteria,  pp.  56-99. 
SCHNEIDER:  Bacteriological  Methods  in  Food  and  Drugs  Laboratories. 

(1915).     Plate  I,  Figs.  2,  3  and  4. 


STUDY  OF  GASEOUS  FERMENTATION  117 


EXERCISE  41.     APPARATUS  AND  METHODS  FOR  THE 
STUDY  OF  GASEOUS  FERMENTATION 

Various  forms  of  yeasts,  bacteria  and  other  micro- 
organisms have  the  ability  to  ferment  carbohydrate,  nitrog- 
enous, and  other  food  substances  with  the  liberation  of  gas. 

A.  Smith's  Fermentation  Tube 

Theobald  Smith  (1893)  introduced  the  use  of  a  special 
tube  for  studying  fermentation  and  gas  production,  and 
now  Smith's  fermentation  tube  is  in  general  use  in  this 
and  other  countries. 

Its  value  lies  in  the  fact  that  it  is  a  simple  apparatus, 
yet  it  allows  not  only  of  testing  the  relative  fermentative 
powers  of  different  species  of  microorganisms  or  of  different 
strains  of  the  same  species,  but  of  determining  the  gases 
produced  qualitatively  and  their  relative  proportions 
quantitatively  to  some  extent. 

Apparatus.  Smith  fermentation  tubes;  gasometer; 
nutrient  carbohydrate  broth  (or  any  desired  solution); 
platinum  needles. 

Culture.     Culture  of  the  organism  to  be  tested. 

Method.  1.  The  carbohydrate  broth  (or  other  liquid 
medium)  is  placed  in  the  fermentation  tubes,  filling  the  long 
arm  by  carefully  tilting.  The  bulb  should  be  filled  with 
the  liquid  only  to  the  extent  that  air  will  not  enter  the  long 
arm  upon  slightly  tilting.  The  tube  should  not  be  filled 
so  full  that  the  bulb  will  not  contain  all  of  the  liquid  in  the 
long  arm. 

2.  Sterilize.     Carbohydrate  broths  are  sterilized  by  the 
intermittent  method. 

3.  Inoculate  fermentation  tubes  of  the  desired  medium 
with  the  organism  to  be  tested,  using  a  loop  or  straight  needle. 

4.  Incubate  at  optimum  temperature. 

5.  Examine  in  twenty-four  hours   for  gas  production, 
and  mark  the  level  of  the  liquid  in  the  long  arm  of  the 


118 


GENERAL  MICROBIOLOGY 


fermentation  tube  each  day  if  gas  is  being  formed.  (If 
the  level  is  higher  than  it  was  the  previous  day,  the  gas 
(CO2)  is  being  absorbed.  Do  not  allow  this  absorption 
to  proceed  further,  but  test  the  gas  present  for  C02 
and  H2). 

6.  Measure  and  record  the  amount  of  gas  by  means  of  a 
gasometer    (see   illustration).     The    total    amount   is   not 


FIG.  36. — Smith's  Fermentation  Tube  Showing  use  of  Frost's  Gas- 
ometer. 


exact  quantitatively,  as  some  gas  is  given  off  from  the 
open  arm  of  the  fermentation  tube. 

7.  When  the  maximum  amount  of  gas  is  formed,  test 
the  gas  for  C02  and  other  gases  as  follows: 

Fill  the  short  arm  of  the  fermentation  tube  with  10% 
NaOH.  Place  the  thumb  over  the  mouth  of  the  tube  and 
shake  vigorously,  so  that  the  gas  contained  in  the  long 
arm  comes  in  contact  with  the  NaOH. 

2NaOH+C02  =  Na2CO3+H2O. 


THE  STUDY  OF  YEASTS  119 

Sodium  carbonate  and  water  are  formed,  leaving  the  other 
gases  free. 

Collect  in  the  long  arm  of  the  tube  all  the  gases  remain- 
ing.  Remove  the  thumb.  The  difference  in  the  per  cent 
of  gas  before  and  after  treating  with  the  NaOH  equals  the 
per  cent  of  CCb  which  was  present. 

8.  Place  the  thumb  over  the  mouth  of  the  tube  and 
collect  all  remaining  gas  in  the  short  arm.  Light  a  match, 
remove  thumb  and  immediately  touch  off  the  remaining  gas. 
If  Eb  is  present  the  typical  reaction  occurs.  Other  gases 
are  often  present,  but  in  too  small  amounts  to  allow  of 
testing. 

10.  Record  the  relative  proportions  of  C02  and  H2 
formed. 

B.  Durham's  Fermentation  Tube 

Durham's  fermentation  tube  is  simply  an  ordinary  test 
tube  containing  a  sugar  broth,  in  which  a  smaller  test  tube, 
inverted,  has  been  placed  before  sterilization. 

This  apparatus  possesses  some  advantages  over  the 
Smith  fermentation  tube  if  only  the  presence  of  gas  produc- 
tion is  to  be  noted,  as  the  tubes  are  more  easily  cleaned, 
sterilized  and  handled. 

The  amount  of  gas  may  be  roughly  estimated,  but  the 
kind  of  gas  may  not  be  determined  by  the  use  of  this 
apparatus, 

EXERCISE  42.     THE  STUDY  OF  YEASTS 

The  object  of  this  exercise  is  to  demonstrate  how 
to  differentiate  yeasts  by  microscopical  and  cultural 
methods. 

Apparatus.  Clean  cover-glasses;  three  clean  concave 
slides;  five  clean  fermentation  tubes;  one  tube  sterile 
2%  dextrose  broth;*  one  tube  sterile  2%  lactose  broth; 

*  The  sugar  and  glycerin  broths  are  furnished  by  the  laboratory. 


120  GENERAL  MICROBIOLOGY 

one  tube  sterile  2%  saccharose  broth;  one  tube  sterile 
2%  glycerin  broth;  one  tube  sterile  nutrient  broth;  three 
tubes  sterile  wort;  three  tubes  sterile  gelatin;  four  tubes 
sterile  dextrose  agar;  gasometer;  10%  NaOH. 

Cultures.  Saccharomyces  cerevisice;*  Saccharomyces 
apiculatus;  Torula  rosea. 

Method.  1.  Fill  one  fermentation  tube  with  each  broth. 
Sterilize  by  heating  one-half  hour  in  the  steam  on  three 
consecutive  days. 

2.  Make  cultures  of  each  yeast  in 
(a)  Beer  wort. 

(6)  Gelatin  (stab  culture). 

(c)  Dextrose  agar  slant. 

(d)  Dextrose  agar  plate  (giant  colony). f 

(e)  Linder's  concave  slide  culture  (p.  83). 

3.  Make    cultures   of  Saccharomyces    cerevisice    only,   in 
fermentation  tubes  of 

(a)  Plain  broth — control — (without  carbohydrate). 

(b)  2%  dextrose  broth. 

(c)  2%  lactose  broth. 

(d)  2%  saccharose  broth. 

(e)  2%  glycerin  broth. 

4.  Prepare  an  adhesion  culture  from  freshly  inoculated 
wort  culture  of  the  yeast  (see  p.  78). 

5.  Examine  microscopically  immediately  after  prepara- 
tion and  draw  single  cells  and  cells  in  various   stages   of 
budding  (germination);   show  interior  structure  of  cells. 

6.  Examine  all  cultures  after  twenty-four  hours,  and  make 
drawings  of  (a),   (b),   (c),   (d),  and  (e)  under  2;   place  as 
indicated  on  chart,  and  label  correctly. 

7.  Describe  all  gelatin  and  agar  cultures  according  to 
the  descriptive  chart  of  the  American  Society  of  Bacteriol- 

*  Saccharomyces  cerevisioe  has  been  previously  isolated  from  a  fresh 
cake  of  Fleischmann's  compressed  yeast.  (See  Exercise  40.) 

t  Only  one  plate  is  necessary.  All  yeasts  may  be  grown  on  one 
plate.  Use  dextrose  agar. 


THE  STUDY  OF  YEASTS  121 

ogists  (p.  134).  In  describing  the  wort  culture  use  the 
descriptive  chart  terms  under  the  heading  "  Nutrient 
broth." 

8.  If  any  gas  has  formed  in  the  fermentation  tubes 
mark    the    level   of    the    liquid    in  the    long  arm  with    a 
wax  pencil  and  record  the  percentage  of    gas,  using   the 
gasometer. 

9.  Test  quantitatively  and  qualitatively  for  gas  in  the 
fermentation  tubes.     (See  Exercise  41,  p.  117). 

10.  What  is  the  ratio  of  the  CO2  to  H2  and  other  gases? 
Is    this    ratio    constant    for    all    fermentations?     For    one 
organism?     Why?     Do  all  organisms  cause  fermentation? 
Why?     What  causes  fermentation? 

11.  Examine  adhesion  cultures  after  forty-eight  hours 
and  seventy-two  hours  and  make  drawing  of  colony  forma- 
tion. 

12.  Study  the  fourteen  to  twenty-day  old  wort  cultures 
in  hanging  drop  for  endospores.      When  do   these  form? 
Why? 

REFERENCES 

HANSEN,  E.:   Practical  Studies  in  Fermentation,  pp.  215-217. 

LAFAR:  Technical   Mycology,   Vol.   II,   Part  II,   pp.   394-406,   430- 

436. 

EYRE:  Bacteriological  Technic.     Second  Ed.,  p.  7. 


122 


GENERAL  MICROBIOLOGY 


YEASTS 


Name  of  student 

Desk  No. 

Name  of  organism 

Isolated  from 

Method  of  isolation 

Occurrence 

Importance 

Total  organism                                Drawn  from  preparation 

Stages  of  budding                             Drawn  from  preparation 

Method  of  reproduction                   Drawn  from  preparation 

Spore 

Fermentations 


%  Of  gas  in 

Control 

Dextrose 

Lactose 

Saccharose 

Glycerin 

24  hours 

48  hours 

3  days 

5  days 

Total  gas 
production 

Ratio  of  CO2:H2 
and  other  gases 

Alcohol  (odor) 

Acid 

Growth  in 
closed  arm 

THE  STUDY  OF  YEASTS 


123 


Iday 

days 

days 

Cider  or  wort 

culture 

Reaction                 . 

Incubated  at 

.   °  C. 

O 

[^ 

[^ 

Iday 

days 

days 

Nutrient  gelatin 

stab 

Reaction    

Incubated  at 

0  C. 

^J 

^J 

U 

Agar  streak 

Reaction 

Incubated  at 

..°C. 


1  day  .days         days 


A 


Age  of  colony 


Size  of  colony 


Surface  elevation 


Gelatin  or  agar 

colony 
Reaction 
Incubated  at 


124 


PLATE  V 


THE   STUDY  OF  BACTERIA  125 


EXERCISE  43.  THE  STUDY  OF  BACTERIA 

Studies  will  be  made  of  ten  bacteria  representing  the 
different  morphological  types.  These  are  to  be  identified 
by  morphological  and  cultural  characteristics. 

Pure  cultures  of  these  organisms  will  be  found  on  each 
desk  in  the  tumblers  marked  "  Laboratory  cultures." 
Always  return  laboratory  cultures  to  these  tumblers  imme- 
diately after  using. 

DANGER.  Some  of  these  organisms  are  pathogenic. 
If  you  do  not  handle  them  with  care  and  according  to 
directions  you  endanger  not  only  yourself,  but  all  working 
in  the  laboratory.  Do  not  be  careless.  Handle  all  organ- 
isms as  if  they  were  pathogenic.  This  is  a  good  habit;  get 
it  immediately.  (See  "  Care  of  Cultures,"  pp.  46-48)  The 
instructor  will  designate  which  organisms  are  pathogenic. 

Apparatus.  Clean  cover-glasses;  clean  concave  slides; 
clean  plain  slides;  ten  agar  slants;  ten  tubes  sterile  agar 
for  plates;  ten  tubes  nutrient  broth;  ten  tubes  nutrient 
gelatin;  ten  tubes  litmus  milk;  ten  tubes  glycerin  potato; 
ten  tubes  Dunham's  solution;  ten  tubes  nitrate  peptone 
solution;  four  fermentation  tubes  of  plain  broth;  four 
fermentation  tubes  of  dextrose  broth;  four  fermentation 
tubes  of  lactose  broth;  four  fermentation  tubes  of  sac- 
charose broth;  centimeter  scale;  gasometer;  lead  acetate 

DESCRIPTION  OF  PLATE  V 

I.  1,  Bad.  tuberculosis;  2,  B.  typhosus;  3,  Bad.  leprce;  4,  Bad. 
anthracis  (strepto-bacterium,  two  with  spores);  5,  Bad.  diph- 
therias (club-shaped);  6,  anthrax  spore,  germinating  (polar); 
7,  B.  amylobader  (clostridium) ;  8,  Streptococcus  pneumonice 
(diplococcus  with  capsule). 

II.  1,  B.  subtilis  (strepto-bacillus,  peritrichous  flagella,  one  with 
spore);  2,  B.  subtilis  (peritrichous  flagella);  3,  formation  of  a 
new  filament  from  a  germinating  spore;  4,  spore  of  B.  subtilis; 
5,  germinating  spore  of  B.  subtilis  (equatorial);  6,  beginning 
germination. 


PLATE  VI 


\     \A 


III.  1,  Spirillum  volutans  (Cohn)  with  lophotrichous  flagella 
(chain  of  three);  2,  Sp.  volutans,  single  cell;  3,  Microspira 
comma,  monotrichous  flagellum;  4,  Spirocheta  obermeieri. 


IV.  1,  Sardna  lutea;    2,  Micrococcus  tetragenus  with  capsule;    3, 
streptococcus;   4,  planococcus;    5,  staphylococcus. 


THE  STUDY  OF  BACTERIA 


127 


paper;     aqueous-alcoholic     fuchsin     and    methylen    blue; 
mordant  for  flagella  stain;   Lugol's  iodin  solution;    anilin- 


FIG.  37. — Cycle  of  Development  of  Bacterial  Cell.     (Adapted  from 
Fuhrmann's  Technische  Mykologie.) 

water  gentian  violet;  carbol-fuchsin;  acetic  acid-alcohol 
for  decolorizing  spore  stain;  indol  test  solutions;  nitrate 
test  solutions;  ammonia  test  solutions. 


128 


GENERAL  MICROBIOLOGY 


Method.  1.  Make  an  agar  slant  culture  of  each  organism 
and  incubate  each  at  its  optimum  temperature.  (Instructor 
will  designate  the  optimum  temperature  of  each.) 


11 


FIG.  38. — Comparative  Sizes  of  Bacteria. 

1,  Micrococcus  progrediens,  0.15^,'  2,  Micrococcus  urea;,  1-1.5^; 
3,  Sarcina  maxima,  4^;  4,  Thiophysavolutans  (sulphur bacteria), 
7-18^;  5,  influenza  bacillus,  4.2X0.4/x;  6,  methane  bacillus, 
5X0.4/*;  7,  Urobacillus  dudauxii  (Miquel),  2-10X0.6-0.8^; 
8,  Bacillus  nitri  (Ambroz),  3-8X2-3^;  9,  Beggialoa  alba 
(sulphur  bacteria),  2. 9-5. 8 X 2. 8-2. 9/*;  10,  Chromatium  okenii, 
(sulphur  bacteria),  10-15X5/*;  11,  Beggiatoa  mirabilis  (sul- 
phur bacteria),  20-25  X40-50/X.  (From  Fuhrmann's  Tech- 
nische  Mykologie.) 

2.  Draw  and  describe  twenty-four-hour  old  agar  slant 
cultures,  then  examine  microscopically  in  hanging  drop  to 
determine  the  morphology,  size,  grouping  or  arrangement, 


THE  STUDY  OP  BACTERIA  129 

motility,  spores.  Use  ocular  No.  1  and  objective  No.  7. 
The  greatest  motility  will  be  observed  in  organisms  growing 
in  the  condensation  water  at  the  base  of  the  slant. 

3.  Draw  the  total  organism  and  record  the  presence  or 
absence  of  motility.     Describe  all  cultures  at  the  time  the 
drawings  are  made  of  each,  following  the  terminology  of 
the  "  Descriptive  Chart  of  the  American  Society  of  Bac- 
teriologists," p.  134. 

4.  Use  drawing  pencil  for  making  drawings  and  ink  for 
recording  descriptions. 

Any  descriptive  terms  may  be  added  which  will  aid  in 
identifying  organisms,  but  descriptive  chart  terms  must 
be  followed  as  closely  as  possible,  otherwise  drawings  will 
not  be  accepted. 

Always  state  the  age  of  the  culture,  the  temperature  at 
which  the  organism  is  grown,  the  medium  upon  which  it  is 
cultivated  and  the  litre  of  the  medium. 

Use  one  chart  for  each  organism. 

5.  When  the  agar  slant  culture  of  each  organism  shows 
good  growth,  make  inoculations  from  this  culture  into  the 
following  media : 

Agar  plate  (see  below  for  method). 

Gelatin  plate  (see  step  6,  below). 

Nutrient  broth. 

Nutrient  gelatin  (stab  culture). 

Litmus  milk. 

Glycerin  potato. 

Dunham's  solution. 

Nitrate  peptone  solution. 

Plain  broth  fermentation  tube  (control). 

Dextrose  broth  fermentation  tube. 

Lactose  broth  fermentation  tube. 

Saccharose  broth  fermentation  tube. 

6.  In  preparing  agar  plate  from  bacterial  cultures,  proceed 
as  follows:   Inoculate  a  tube  of  nutrient  broth  lightly,  using 


130  GENERAL  MICROBIOLOGY 

the  straight  needle.  Then,  still  using  the  straight  needle, 
from  the  freshly  prepared  broth  culture,  inoculate  lightly 
one  tube  of  melted  agar  (at  40°  to  50°  C.)  and  pour  into  a 
sterile  Petri  dish.  If  the  organism  shows  only  a  slight  growth 
on  the  stock  culture,  transfer  directly  to  melted  agar. 

7.  Moisten  a  strip  of  lead  acetate  paper  and  insert  with 
cotton  plug  in  tube  of  Dunham's  solution.     Blackening  of 
this  paper  shows  the  formation  of  H2S. 

Between  what  substances  does  a  chemical  reaction  take 
place?  What  are  the  resulting  products? 

8.  Draw  and  describe  twenty-four-hour  cultures  of  the 
first  four  bacteria  in  all  media.     If  at  any  time  presence 
of  growth  is  doubtful,   compare    with    a    tube  .  of    sterile 
medium.     In  the  absence  of  growth,  reinoculate. 

9.  Record  macroscopical  changes  only,  in  litmus  milk; 
and  in  fermentation  tubes  note  only,  the  place  of  growth, 
presence  and  percentage  of  gas;   also  the  formation  of  H2S 
in  Dunham's  solution. 

10.  Make  a  permanent  stained  preparation  of  each  organ- 
ism    (following    directions    under    Exercise    28).     Young 
(twenty-four  to   forty-eight  hour)    cultures   must    be    used. 
Use  either  aqueous-alcoholic  fuchsin  or  aqueous-alcoholic 
methylen  blue. 

11.  Make  a  flagella  stain  of  the  largest  motile  organism 
among  your  cultures. 

It  is  absolutely  necessary  that  a  young  (eighteen  to  twenty- 
four  hour,  not  older)  culture  be  used  for  this  purpose.  Fol- 
low the  directions  under  Exercise  31. 

12.  Make  further  drawings  and  descriptions  from  day 
to  day  if  any  change  in  the  growth  from  that  of  the  preceding 
day  is  observed.     Three  drawings  of  a  culture  will  be  suf- 
ficient.    Endeavor    to    illustrate    typical    growth    by    careful 
drawings. 

13.  State  whether  the  agar  plate  colony  described  is  a 
surface  or  a  subsurface  colony.     How  do  these  two  types  of 
colonies  differ?     Why? 


THE  STUDY  OF  BACTERIA  131 

14.  Note  the  presence  of  condensation  water,  whether 
a  small  or  large  amount  is  present.     How  does  this  affect 
colony  development? 

15.  Draw  and  measure  a  typical  surface  and  subsurface 
colony  produced  by  each  organism. 

The  form  and  size  often  vary  with  the  physical  con- 
dition under  which  the  colony  grows  or  with  physiological 
conditions,  i.e.,  the  proximity  of  colonies  producing  poison- 
ous metabolic  products. 

16.  Examine  cultures  three  to  six  days  old  in  hanging 
drop  for  presence  of  spores.     Spores  may  be  seen  free  or 
enclosed  in  the  bacterial  cells.     They  are  easily  distinguished 
by  their  refractivity.     Ordinary  anilin  dyes  will  not  stain 
them. 

17.  Make    a   contrast    spore    stain    of    a    spore-forming 
organism.     (For  method  see  Exercise  29.) 

Draw  and  describe  only  the  mature  cultures  of  the  last 
six  organisms  (five  to  eight  days  old) . 

18.  Make  the  indol,   nitrate  and  ammonia  tests  also 
on  the  mature  cultures. 

19.  In  fermentation  tube  cultures  note  and  record  the 
oxygen  requirements  of  each  organism;   total  per  cent  of 
gas;   ratio  of  CO2  :  H2  and  other  gases. 

20.  Test   each   organism    after   seven  days    for   indol, 
nitrate  and  ammonia   production.     The   culture  in  Dun- 
ham's peptone   solution  is  tested   for  indol    (for  method 
see  Exercise  44). 

Divide  the  nitrate  peptone  solution  culture  into  two  parts; 
test  one  for  nitrates,  the  other  for  ammonia  (for  method 
see  Exercise  45). 

21.  Prepare    permanent    stained    preparations    of    one 
Gram-positive  and  one  Gram-negative  organism. 

22.  Making  use  of  morphological  and  cultural  charac- 
teristics   ascertained    microscopically  and    by  the   various 
cultural    tests,    identify    each    organism,    using    Chester's 
Manual    of    Determinative    Bacteriology  for   tracing    out 


132  GENERAL  MICROBIOLOGY 

the  genus  and   species.      Other   valuable   reference    texts 
are: 

CONN,  ESTEN  and  STOCKING:  Classification  of  Dairy  Bacteria. 
NOVY:  Laboratory  Manual  of  Bacteriology. 
JORDAN:  General  Bacteriology. 

EXERCISE  44.     EHRLICH'S  METHOD  OF  TESTING 
INDOL  PRODUCTION 

The  purpose  of  the  exercise  is  to  test  the  power  of  an 
organism  to  produce  indol  from  peptone. 

Cultures  for  comparison  should  be  of  the  same  age  and 
grown  in  the  same  kind  of  medium.  Some  peptones  con- 
tain a  trace  of  indol  and,  to  avoid  all  possibility  of  mis- 
take when  testing  for  indol,  a  control  tube  of  sterile  medium 
should  be  used  at  the  same  time.  This  reaction  is  char- 
acteristic for  indol  or  for  methyl  indol  (skatol). 

There  are  other  tests  for  indol,  but  this  one  is  by  far 
the  most  delicate.  The  Salkowski-Kitasato  test  (cone. 
H2SO4  and  NaNCb)  will  detect  indol  in  a  dilution  of  only 
1  :  100,000,  while  Ehrlich's  test  will  give  a  reaction  in  a 
dilution  ten  times  greater,  or  1  :  1,000,000. 

Indol  is  one  of  the  most  important  of  protein  decom- 
position -  products.  It  is  noted  for  its  intense  fecal  odor. 
However,  in  highly  dilute  solutions  it  has  the  odor  of  orange- 
blossoms,  hence  is  used  extensively  in  perfumery.  The 
jessamine  blossom  contains  indol  and  has  its  odor. 

Indol  has  the  following  graphic  formula: 

H 


UC/      C CH 

I        D      II 


H 


According  to  Emil  Fischer,   the  reaction  of  Ehrlich's 
test,  produces,  by  means  of  the  oxidizing  action  of  the  potas- 


TESTS  FOR  THE  REDUCTION  OF  NITRATES      133 

sium  persulphate,  a  condensation  of  two  molecules  of  indol 
with  the  aldehyde  group  of  the  para-dimethyl-amido- 
benzaldehyde,  water  splitting  off. 

Apparatus.  Solutions  I  and  II  for  Ehrlich's  test  for 
indol;*  two  clean  5  c.c.  pipettes. 

Culture.  Dunham's  peptone  solution  or  broth  culture 
of  the  organism  to  be  tested. 

Method.  1.  To  about  10  c.c.  of  the  liquid  culture 
add  5  c.c.  of  solution  I,  then  5  c.c.  of  solution  II. 

2.  Shake  the  mixture.  The  reaction  may  be  accelerated 
by  heating.  The  presence  of  indol  is  indicated  in  a  few 
minutes  by  a  red  color  which  increases  in  intensity  with 
time.  For  standard  compaiisons,  five  minutes  is  taken 
as  the  maximum  time  limit. 

REFERENCES 

BOEHME,  A.:    Die  Anwendung  der  Ehrlichschen  Indol-reaktion  fur 

Bakteriologische  Zwecke.      Cent.  f.  Bakt.  Orig.  Bd.  40  (1906), 

pp.  129-133. 
BESSON:    Practical  Bacteriology,   Microbiology  and  Serum  Therapy 

(1913),  p.  374. 

LOHNIS:  Laboratory  Methods  in  Agricultural  Bacteriology  (1913),  p. 42. 
LEWIS,  F.  C.:  On  the  detection  and  estimation  of  bacterial  indol  and 

observations  on  intercurrent  phenomena.     Jour.  Path,  and  Bact., 

Vol.  19  (1915),  pp.  429-443. 

EXERCISE  45.    TESTS  FOR   THE   REDUCTION    OF 
NITRATES 

The  purpose  of  the  exercise  is  to  test  the  power  of  an 
organism  to  reduce  nitrates. 

Apparatus.  Sulphanilic  acid,  nitrite  test  solution  I; 
a-naphthylamin,  nitrite  test  solution  II;  Nessler's  solution; 
phenolsulphonic  acid. 

Cultures.  Seven-day  old  nitrate  peptone  solution  cul- 
tures grown  at  20°  to  25°  C.,  or  four-day  old  nitrate  pep- 
tone solution  cultures  (pathogenic)  grown  at  37°  C. 

Method.     (A)  For   nitrites:     1.  Add    0.1    c.c.    each    of 
solutions  I  and  II  to  each  culture  to  be  tested. 
*  See  Appendix. 


134  GENERAL  MICROBIOLOGY 

2.  Repeat  with  uninoculated  control. 

3.  The  development  of  a  red  color  in  ten  minutes  indi- 
cates the  presence  of  nitrites,  the  intensity  of  the  color 
depending  upon  the  amount  of  nitrites  present. 

(B)  For  ammonia.     1.  Add  0.2  c.c.  of   Nessler's    solu- 
tion to  each  culture  to  be  tested. 

2.  Repeat  with  uninoculated  control. 
The  presence  of  ammonia   is  shown  by  a  yellow  color 
or  precipitate. 

(C)  For   nitrates   unchanged   or  free   nitrogen   liberated. 
1.  When  either  or  both  of  the  preceding  tests  are  positive, 
no  further  determination  need  be  made,  but  if  negative, 
then  one  of  two  conditions  may  prevail:     (a)  Either  the 
nitrates  have  not  been  changed,  or  (6)  they  may  have  been 
reduced  to  free  nitrogen.     To   ascertain  which  is  true,  it 
will  be  necessary  to  determine  the  presence  or   absence 
of  nitrates. 

2.  Test  as  follows:  (a)  Evaporate  10  c.c. 'of  each  cul- 
ture and  the  controls  almost  to  dryness  in  an  evaporat- 
ing dish  and  add  to  the  residue  1  c.c.  of  phenolsulphonic 
acid. 

(6)  Dilute  with  10  c.c.  distilled  water,  then  add  suf- 
ficient ammonium  hydroxide,  diluted  1  :  1  with  distilled 
water,  or  concentrated  potassium  hydroxide  solution, 
to  make  alkaline. 

(c)  Transfer  the  liquid  to  a  50  c.c.  Nessler  tube  or  grad- 
uated cylinder  and  make  up  the  volume  to  50  c.c.  with 
distilled,  water. 

A  yellow  color  shows  the  presence  of  nitrates. 


TESTS  FOR  THE  REDUCTION  OF  NITRATES       135 
BACTERIA 


Name  of  student 

Desk  No. 

Name  of  organism                                      Isolated  from 

Method  of  isolation 

Occurrence 

Importance 

Shape  of  organism 

Arrangement 

Size 

Motility 

Flagella 

Method  of  reproduction 

Involution  forms 

Spore 

Stages  of  germination 

Aqueous-alcoholic  stain              Gram's  stain               Aci 

d-fast  stain 

1  day  days         days 


Agar  streak 

Titre 

Incubated  at 


1  day  days          days 


Gelatin  stab 

Titre 

Incubated  at 


C. 


136 


GENERAL  MICROBIOLOGY 

1  day  days  days 


Broth  culture 
Titre 
Incubated  at 


Potato  culture 
Titre  ........... 

Incubated  at 
............  °  C. 


1  day  days         days 


v 


Age  of  agar  colony 

days 

days 

days 

Size  of  colony 

Surface  elevation 

Agar  colony 
Titre  .....  . 

Incubated  at 


TESTS  FOR  THE  REDUCTION  OF  NITRATES       137 


Age  of  gelatin  colony 

days 

days 

days 

Size  of  colony 

Surface  elevation 

Gelatin  colony 
Titre    

Incubated  at 
°C. 

Litmus  milk 

Acid 

Gas 

Acid  curd 

Rennet  curd 

Reduction 

Alkali 

Peptonization 

Fermentations 


%  Of  gas  in 

Control 

Dextrose 

Lactose 

Saccharose 

Glycerin 

24  hours 

48  hours 

3  days 

7  days 

Total  gas 
production 

Ratio  of  CO2:H2 
and  other  gases 

Acid 

Growth  in 
closed  arm 

138 


GENERAL  MICROBIOLOGY 


Chromogenesis 
on 

Nutrient  broth 

Nutrient  gelatin 

Nutrient  agar 

Potato 

Production  of 

NH3  from  peptone 

H^S  from  peptone 

Indol  from  peptone 

Nitrites  from  peptone 

Reduction  of 
nitrates  to 

NH3 

Nitrites 

Remarks: 


EFFICIENCY  OF  INTERMITTENT  HEATING       139 

EXERCISE  46.  TO  DEMONSTRATE  THE  EFFICIENCY 
OF  INTERMITTENT  HEATING  AS  A  METHOD  OF 
STERILIZING  MEDIA.  ALSO  TO  COMPARE  THE 
EFFICIENCY  OF  CONTINUOUS  AND  INTERMITTENT 
HEATING 

Apparatus.  400  c.c.  fresh  skim  milk;  forty  sterile 
test  tubes;  2%  azolitmin  solution. 

Method.  1.  Prepare  litmus  milk  according  to  direc- 
tions on  p.  25. 

2.  Fill  the  tubes,  using  approximately  8  c.c.  per  tube. 

3.  Set  five  away  without  heating. 

Heat  five  for  fifteen  minutes  on  the  first  day. 
Heat  five  for  one  hour  on  the  first  day. 
Heat  five  for  fifteen  minutes  on  two  successive  days. 
Heat  five  for  fifteen  minutes  on  three  successive  days. 
Heat  five  for  fifteen  minutes  on  four  successive  days. 
Heat  ten  for  fifteen  minutes  on  five  successive  days. 

4.  Keep    all    tubes    at    room    temperature.     Examine 
every  two  or  three  days  and  describe  the  macroscopical 
changes  of  each  set,  as  described  under  the  discussion  on 
litmus  milk  (pp.  23-25.) 

Why  do  not  all  the  tubes  of  a  set  change  alike?  Why 
do  not  all  sets  present  the  same  appearance? 

Save  all  tubes  that  do  not  show  macroscopical  changes. 
These  are  probably  sterile. 

6.  Tabulate  your  results  after  ten  days  to  two  weeks, 
recording  the  number  and  per  cent  of  each  lot  that  shows 
macroscopical  changes. 

6.  Is  milk  difficult  to  sterilize?  Why?   What  other  media 
present  the  same  problem  of  sterilization  as  milk?     Why? 

Would  any  other  method  for  the  sterilization  of  milk  be 
preferable  to  the  ones  you  used?  Give  reasons  for  your 
answer. 

7.  State  your  results  in  detail  and  point  out  any  con- 
clusions that  may  be  drawn  and  any  practical  applications 
that  may  be  made. 


140  GENERAL  MICROBIOLOGY 


REFERENCES 

MARSHALL:  Microbiology,  pp.  153-161,  306-313,  363-365. 
CONN,  H.  W.:  Bacteria,  Yeasts  and  Molds,  pp.  191-193. 
BESSON,  A.:  Practical  Bacteriology,  Microbiology  and  Serum  Therapy, 
pp.  35-36. 


EXERCISE  47.     TO  COMPARE  MORPHOLOGICALLY 
PROTOZOA  WITH  BACTERIA 

Apparatus.  Deep  culture  dish;  concave  slide;  clean 
cover-glasses;  cover-glass  forceps;  platinum  loop;  tube 
of  sterile  broth;  tube  of  sterile  Chinese  ink. 

Cultures.     Rich  soil  or  slimy  leaves  from  a  pond. 

Method.  1.  Place  the  soil  or  leaves  in  the  deep  culture 
dish. 

2.  Fill   the   dish   two-thirds   full   with   tap   water   and 
add  the  contents  of  a  tube  of  broth. 

3.  Keep    the    dish    at   room   temperature   for   twenty- 
four  to  forty-eight  hours. 

4.  At  the  end  of  the  incubation  period,  make  a  hanging 
drop  from  the  supernatant  liquid.     Before  inverting  the 
drop  on  the  slide,  add  to  it  that  amount  of  Chinese  ink 
that  adheres  to  the  end  of  a  platinum  needle. 

By  the  use  of  this  ink,  organisms  are  brought  out  by 
contrast,  showing  white  on  a  dark  field.  The  organisms 
are  not  killed  or  injured  by  the  ink. 

5.  Observe  and  measure  any  protozoa,  using  the  lowest 
power  objective  with  the  step  micrometer.     Record  the  size 
in  micra. 

6.  Roughly  sketch  the  different  species  observed,  giving 
comparative  measurements. 

7.  Using  the  highest  power  dry  objective,  observe  bac- 
teria, noting  morphology  and  size. 

8.  Draw  lines  to  represent  the  ratio  between  the  size 
of  the  predominant  types  of  each. 

9.  Are  the  protozoa  present  visible  to  the  naked  eye? 


THE  DECOMPOSITION  OF  MILK  141 

How  many  of  the  largest  protozoa  present,  placed  end  to 
end,  would  make  an  inch? 

10.  How   do   the   protozoa   and   bacteria  in   the   drop 
compare  in  numbers?     Do  these  organisms  have  any  rela- 
tion to  each  other?     If  so,  explain. 

11.  Of  what  importance  are  protozoa?     Name  several 
well-known  protozoa. 

12.  State  your  results  in  detail  and  point  out  any  con- 
clusions that  may  be  drawn  and  any  practical  applications 
that  may  be  made. 

REFERENCE 

MARSHALL:  Microbiology,  pp.  10-11,  68-80,  82-84. 

EXERCISE  48.     TO  STUDY  THE  NATURAL  DECOMPO- 
SITION OF  MILK 

Apparatus.  500  c.c.  sterile  Erlenmeyer  flask;  two 
5  c.c.  sterile  pipettes;  ten  1  c.c.  sterile  pipettes;  four 
10  c.c.  sterile  pipettes;  six  200  c.c.  sterile  Erlenmeyer 
flasks;  fifteen  sterile  Petri  dishes;  physiological  salt  solu- 
tion. 

Cultures.     Fresh  skim  milk. 

Method.  1.  Prepare  "  dilution  flasks "  as  given  in 
Exercise  13,  p.  52,  making  two  90  c.c.  and  four  99  c.c. 
flasks.  Sterilize  by  heating  one  hour  in  flowing  steam  or 
five  minutes  in  the  autoclav  at  120°  C.  (15  Ibs.  pressure). 
Dilution  flasks  and  all  glassware  must  be  sterile  before  the 
experiment  proper  can  be  started. 

2.  Place  200  c.c.  of  the  fresh  skim  milk  in  the  sterile 
500  c.c.  flask  and  use  this  sample  for  the  entire  experi- 
ment. 

3.  Plate  the  milk  immediately  on  nutrient  agar,  using 
dilutions   according  to  the   age  of    the   milk,    as   follows. 
(See   Exercise   13,   p.   52,   for   method    of    using  dilution 
flasks.) 


142  GENERAL  MICROBIOLOGY 

Age.  Dilutions. 

Fresh  milk.  . 1  :  1,000,  1  :  10,000  and  1  :  100,000 

One  day  old 1  :  10,000, 1  :  100,000  and  1  :  1  M  * 

Four  days  old 1  :  10  M,  1  :  100  M  and  1  :  1,000  M 

Eight  days  old 1  :  10  M,  1  :  100  M  and  1  :  1,000  M 

Ten  days  old 1  :  1  M,  1  :  10  M  and  1  :  100  M 

Keep  the  plates  at  room  temperature. 

Sterile  pipettes  are  to  be  used  always  in  making  dilu- 
tions, plating  and  titrating. 

After  the  milk  curdles  it  is  advised  to  make  the  first 
dilution  1  :  10  to  give  a  more  uniform  sample,  from  which 
further  dilutions  are  made.  Use  a  10  c.c.  pipette  having 
a  large  opening  in  the  delivery  end  to  prevent  clogging. 

4.  Titrate  the  milk  sample  every  day.     After  the  milk 
curdles,   shake   well    before  titrating   and  choose  a  5  c.c. 
pipette  having  a  large  aperture  for  delivery  for  obtaining 
the  sample  for  titration. 

5.  Record  the  reaction  in  degrees  of  Fuller's  scale. 
After  using  pipettes,  dilution  flasks,  etc.,  clean,  refill 

and  sterilize  them  at  once  for  future  use. 

6.  Note  the  macroscopical  changes  in  the  milk  sample 
(due  to  microbial  growth),   e.g.,  kind  and  consistency  of 
curd,  extrusion  of  whey,  gas  formation,  peptonization;  also 
note  odor  from  time  to  time. 

7.  Note  the  macroscopical  evidences  of  microbial  growth 
such  as  molds,  etc.,  and  the  time  of  appearance.     Identify 
the  group  to  which  these  organisms  belong,  giving  genus 
and  species  if  possible. 

8.  Determine   the   changes  in  the   numbers   of  micro- 
organisms by  counting  the  colonies  of  the  different  sets  of 
plates  after  they  have  developed  seven  days  at  room  tem- 
perature (see  p.  56,  Exercise  14,  for  method). 

9.  Estimate  the  number  of  colonies  of  each  type  (see 
Exercise  14,  p.  56). 

*M  =  Million. 


THE  DECOMPOSITION  OF  MILK 


143 


10.  Record  your  results,  noting  the  date  on  which  the 
plates  were  made,  the  age  of  the  milk,  the  dilution,  number 
of  colonies  on  the  plate  and  the  average  number  of  organisms 
per  cubic  centimeter. 

11.  Examine  in  hanging  drop  and  note  the  morphology 
of  the   microorganisms   producing  the   most   predominant 
types  of  colonies  on  each  set  of  plates.     Indicate  after  the 
drawing,  the  comparative  numbers  on  each  set  of  plates 
by  the  signs  — ,  +  — ,  +,  +  +  ,  etc.,  to  indicate  absence, 
presence  of  few,  or  many  of  the  type. 

12.  Note  whether  molds  or  yeasts  are  present  on  any 
set  of  plates.     Should  either  be  found  on  fresh  milk  plates? 
Why?     What  types  of  microorganisms  would  you  expect 
on  fresh  milk  plates? 

13.  Prepare  your  data  according  to  the  following  dia- 
gram: 


Date 

Age 

Acidity 

Dilution 

Count 
per  cc. 

Organisms 

Types 

Relative  Nos. 

Feb.  2 

Fresh 
(26  hrs.  old) 

+  15° 

1-1,000 
1-10,000 
1-100,000 

278,900 
325,500 
300,000 

Acid 
Yellow 

+_ 

Average  count  per  cc 301,470 

14.  Plot  the  curve  showing  the  change  in  acidity  and  one 
illustrating  the  count  per  cubic  centimeter  on  the  same 
paper,   using  different  colored  inks  or  different  types  of 
lines.     Use  days  for  abscissae,  acidity  and  count  for  ordinates. 
Start  at  the  same  origin. 

15.  Is  there  any  relation  between  the  change  in  acidity 
and  the  change  in  flora? 

Should  the  acidity  and  count  curves  run  parallel?  If 
they  do  not,  give  a  reason  why. 

How  could  the  bacterial  count  be  made  to  increase 
after  it  goes  down  to  a  constant  number? 


144  GENERAL  MICROBIOLOGY 

16.  What    biochemical   changes    have   occurred   in   the 
decomposition? 

17.  Compare  the  flora  of  fresh  milk,  70°  acid  milk  and 
ten-day  milk,  both  microscopically  and  from   the  plates. 
Explain. 

„  18.  State  your  results  in  detail  and  give  any  conclusions 
to  be  drawn  and  any  practical  applications  that  may  be 
made. 

REFERENCES 

CONN:  Practical  Dairy  Bacteriology,  pp.  21-57,  81-85. 
MARSHALL:  Microbiology,  pp.  298-299,  306-313,  321-326. 

EXERCISE  49.     TO     ISOLATE     SPORE-FORMING    BAC- 
TERIA AND  TO  STUDY  SPORE    FORMATION 

Apparatus.  Two  tubes  of  sterile  broth;  small  piece 
of  hay;  three  sterile  Fetri  dishes;  clean  test  tube;  three 
tubes  sterile  agar  for  plates;  three  sterile  agar  slants; 
carbol-fuchsin;  acetic  acid  alcohol;  aqueous-alcoholic 
methylen  blue;  platinum  needle  and  loop;  ordinary  for- 
ceps. 

Culture.     Hay. 

Method.  1.  Place  a  piece  of  hay  in  the  clean  test 
tube,  plug  the  tube,  and  sterilize  in  the  hot-air  oven. 

2.  Using  sterile  forceps,  place  the  sterile  hay  "  aseptic- 
ally  "  in  one  tube  of  broth  and  an  unsterilized  piece  in  the 
other. 

3.  Incubate  both  at  room  temperature  for  forty-eight 
hours.     Do  both  tubes  show  growth? 

4.  Heat  in  a  water-bath  at  80°  C.  for  ten  minutes  the 
tube  which  shows  marked  growth.     What  does  this  accom- 
plish? 

5.  Make  three  loop-dilution  plates  from  the  heated  broth 
tube. 

6.  Place  the  plates  at  room  temperature  and  examine 
them  daily  for  colony  development. 


REMOVING  MICROORGANISMS   FROM  LIQUIDS     145 

7.  Make  pure  cultures  on  agar  slants  from  three  differ- 
ent well-isolated  colonies  of  the  predominant  types  and  in- 
cubate at  room  temperature. 

8.  Examine    these    in    thirty-six    to    forty-eight    hours 
in  a  hanging  drop  for  morphology  and  spore  formation. 

9.  Make  a  spore  stain  as  soon  as  spores  are  found. 
Where  are  the  spores  located  in  the  bacterial  cell? 

10.  Have  you  studied  any  pure  culture  of  bacteria  which 
is  similar  to  the  types  you  have  isolated?    What  organism 
is  commonly  found  in  hay?     In  what  form  does  it  exist  on 
the  hay?    What  do  you  know  of  the  habitat  of  this  organ- 
ism and  related  forms?     Of  the  pathogenicity? 

11.  State  the  results  obtained  in  detail;    draw  the  con- 
clusions which  follow  and  point  out  any  possible  practical 
applications. 

REFERENCES 

MARSHALL:  Microbiology,  pp.  5,  45,  154,  189,  242. 
JORDAN:  General  Bacteriology,  4th  Ed.,  pp.  235-236. 
EYRE:  Bacteriological  Technic,  2d  Ed.,  p.  140. 

EXERCISE  50.  TO  DEMONSTRATE  THE  EFFICIENCY 
OF  FILTRATION  AS  A  MEANS  OF  REMOVING 
MICROORGANISMS  FROM  LIQUIDS 

Apparatus.  Six  small  funnels;  two  small  filter  papers; 
two  small  pieces  of  absorbent  cotton;  two  small  pieces  of 
clean  hospital  gauze;  eight  tubes  sterile  agar;  eight  sterile 
Petri  dishes;  ten  sterile  1  c.c.  pipettes;  sterile  10  c.c. 
pipette;  three  dilution  flasks;  six  sterile  test  tubes;  tube 
of  sterile  broth. 

Culture.    B.  coli. 

Method.  1.  Inoculate  broth  with  B.  coli  and  incubate 
for  twenty-four  hours  at  21°  C. 

2.  Sterilize  filter  paper  in  each  of  the  two  small  funnels, 
a  small  piece  of  absorbent  cotton  in  each  of  two  more; 
fold  two  pieces  of  clean  gauze  several  thicknesses  and 


146  GENERAL  MICROBIOLOGY 

sterilize  in  the  remaining  two  funnels.     Wrap  all  in  paper 
and  sterilize  in  the  hot-air  oven. 

3.  Shake  the  broth  culture  of  B.  coli  and   plate,  using 
dilutions  1  :  1,000  and  1  :  10,000. 

4.  Filter  each  dilution  (1  :  1,000  and  1  :  10,000)  through 
each  of  the  different  substances,   catching  the  filtrate  in 
sterile  test  tubes. 

5.  Plate  1  c.c.  from  each  filtrate  immediately  and  incu- 
bate the  plates  at  21°  C. 

6.  At  the  end  of  five  days,  count  the  plates. 

7.  Which  method  of  filtration  is  most  efficient?     Why? 
What  factors  could  greatly  influence  the  numbers  of  micro- 
organisms developing  on  the  plates  after  filtration? 

8.  What  methods  are  most  efficient  in  removing  micro- 
organisms from  liquids?     Why? 

9.  Suggest   some   natural   methods   of   filtering   micro- 
organisms. 

10.  Give  in  detail  the  results  obtained,  state  any  con- 
clusions that  may  be  drawn  and  point  out  any  practical 
applications. 

REFERENCES 

EYRE:  Bacteriological  Technic,  2d  Ed.,  pp.  42-48. 
MARSHALL:  Microbiology,  pp.  64-67. 

EXERCISE  61.     TO  DEMONSTRATE  PRESENCE  OF  MI- 
CROORGANISMS IN  AIR,  ON  DESK,  FLOOR,  ETC. 

Apparatus.     Six  sterile  Petri  dishes;   six  tubes  of  sterile 
agar. 

1.  Air.    Method.     1.  Pour  six  plates  with  uninoculated 
sterile  agar  and  set  on  a  level  surface  until  solid. 

2.  Expose  one  plate  for  one  minute  to   (a)   laboratory 
air;    (6)  air  of  campus;    (c)  air  of  your  room  while  sweep- 
ing or  dusting. 

II.  Floor.     1.  Bend   the   straight   platinum   needle   tilf 
it  forms  a  right  angle. 


PRESENCE  OF  MICROORGANISMS  IN  AIR,  ETC.  147 

2.  Sterilize  it  in  the  flame. 

3.  Moisten  the  needle  with  sterile  water. 

4.  Rub  it  along  the  floor,  and  then, 

5.  Draw  it  lightly  across  the  surface  of  the  agar  in  the 
fourth  Petri  dish. 

III.  Desk.  1.  Sterilize  the  needle  and  repeat  operation 
(II,  5)  obtaining  the  inoculum  from  the  surface  of  a  desk 
which  has  not  just  previously  been  washed  with  1  :  1,000 
mercuric  chloride. 

2.  Then  wash  the  surface  of  the  desk  well  with  this 
solution  and  when  the  desk  top  is  dry,  repeat  the  operation, 
using  the  sixth  plate. 

3.  Mark  all  plates  with  the  date  on  which  they  were 
exposed  or  inoculated  and  place  them  at  a  constant  tem- 
perature. 

4.  Watch  any  developments  from  day  to  day.     What 
organisms  predominate  on  the  plates?    Why? 

5.  Examine  different  colonies  in  a  hanging  drop.     What 
types  of  bacteria  are  found? 

6.  Upon  what  does  the  species  and  number  of  micro- 
organisms depend?     What  becomes  of  them  when  air  cur- 
rents are  present?     When  the  floor  is  swept  in  the  ordinary 
way?     Mopped?    When  the  desk  is  washed  with  water? 
With  mercuric  chloride? 

7.  Are  these  types  deleterious  to  health?    Why  should 
and  how  may  they  be  avoided  in  the  laboratory?     Out- 
side of  the  laboratory? 

8.  State  your  results  for  I,  II  and  III  in  detail,  draw  any 
conclusion  possible  and  point  out  any  practical  operations. 

REFERENCES 

MARSHALL:  Microbiology,  pp.  185-191. 

BESSON:    Practical  Bacteriology,  Microbiology  and  Serum  Therapy, 

pp.  862-863. 

CONN:  Bacteria,  Yeasts  and  Molds,  pp.  114-123. 
CONN:  Practical  Dairy  Bacteriology,  pp.  65-67. 
TYNDALL:  Floating  Matter  of  the  Air. 


148  GENERAL  MICROBIOLOGY 


EXERCISE  52.  QUALITATIVE  STUDY  OF  THE  MICRO- 
FLORA  OF  THE  SKIN  AND  HAIR  IN  HEALTH  AND 
IN  DISEASE 

Apparatus.  Ordinary  forceps;  two  sterile,  small  white 
enamel  basins  (steam-sterilized);  three  sterile  1  c.c.  pipettes; 
six  sterile  Petri  dishes;  six  tubes  of  sterile  agar;  one  liter 
flask  containing  about  700  c.c.  of  sterile  water  or  salt 
solution;  sterile  cloth  (J  yd.  hospital  gauze  wrapped  in 
paper  and  sterilized  at  180°  C.);  soap;  clean  slides  and 
cover-glasses. 

Method.  I.  Skin,  (a)  Normal.  1.  Place  about  half 
the  sterile  water  in  a  sterile  basin. 

2.  Wash  the  hands  thoroughly  in  the  sterile  water. 

3.  Plate  1  c.c.  of  this  water  immediately  in    ordinary 
agar. 

4.  Then  wash  the  hands  well  with  soap  and  tap  water, 
rinse  with  tap  water  till  free  of  soap  and  dry  the  hands  on 
the  sterile  cloth. 

5.  Place    the    remaining    sterile    water    in    the    second 
sterile  basin  and  wash  the  hands  again. 

6.  Plate  1  c.c.  of  this  water. 

7.  Incubate  both  plates  (inverted)  at  37°  C. 

8.  How  long  before  starting  this  experiment  did  you 
wash  your  hands?     How  might  this  influence  your  results? 

9.  What   types   of   microorganisms   would   you   expect 
to  find  on  the  skin?     Why? 

(6)  Diseased.  1.  With  a  sterile  needle  obtain  a  small 
amount  of  purulent  material  from  a  pustule,  boil,  or  abscess, 
etc. 

2.  Make  three  loop-dilution  plates  in  agar.      Incubate 
at  37°  C. 

3.  Examine  some  of  this  material  microscopically  by 
preparing  a  stained  smear. 

4.  Draw   and    describe   the    latter.     Do   you   find    the 
same  organisms  on  the  plates  as  on  the  slide? 


MICROFLORA  OF  THE  SKIN  AND  HAIR  149 

6.  Isolate  the  most  predominant  organism  on  the  plates 
and  identify  them. 

6.  What   is   the   source   of   all   these   microorganisms? 
What  becomes  of  them  when  we  wash  our  hands  and  wipe 
them  in  the  ordinary  way?    Are  they  detrimental  to  health? 

7.  What  is  pus?     Of  what  does  it  consist?    What  care 
should  be  taken  with  discharges  from  suppurating  sores? 

II.  Hair,  (a)  Normal.  1.  Using  flame-sterilized  forceps 
(ordinary  type),  obtain  several  hairs  and  place  them  in  a 
sterile  Petri  dish. 

2.  Using  a  sterile  pipette,  add  1  c.c.  of  sterile  water 
or  salt  solution  to  the  Petri  dish  and  stir  the  hairs  about 
in  it  with  the  pipette  or  sterile  loop  in  order  to  dislodge  the 
organisms  adhering  to  them. 

3.  Pour  into  the  plate  a  tube  of  melted  agar  (at  40° 
to  45°  C.),  and  when  hard,  incubate  at  37°  C. 

4.  After    twenty-four    to    forty-eight    hours,    examine 
predominating  colonies  in  a  hanging  drop. 

(b)  Diseased.  1.  With  sterile  forceps  obtain  a  few  hairs 
from  the  growing  edge  of  the  infected  portion  of  the  skin 
affected  with  ringworm  or  barber's  itch.  These  .hairs  will 
come  out  easily  in  comparison  with  healthy  hairs. 

2.  Mount  and   examine    for    the  fungus,   Trichophyton 
tonsurans.      (See     illustration    on    p.    578    in    Marshall's 
Microbiology.)     Draw. 

3.  Continuous    application    of   a    glycerinated    solution 
of  1  :  500  HgCl2  (glycerin  1  part,  HgCl2   1  :  500,  9  parts) 
will  kill  this  fungus. 

State  in  detail  your  results  for  I  and  II,  draw  any  con- 
clusion permissible  and  point  out  any  practical  application. 

REFERENCES 

MARSHALL:  Microbiology,  pp.  522-524,  545,  578,  591-593. 
BESSON:    Practical  Bacteriology,  Microbiology  aud  Serum  Therapy, 
pp.  679-688. 


150  GENERAL  MICROBIOLOGY 


EXERCISE  63.  QUALITATIVE  STUDY  OF  THE  MICRO- 
FLORA  OF  THE  MUCOUS  MEMBRANE  (MOUTH 
AND  THROAT  OR  NOSE) 

Apparatus.  Absorbent  cotton,  small  piece;  wire  rod 
about  15  cms.  long;  clean  slides  and  cover-glasses;  Petri 
dish,  sterile;  tube  of  sterile  agar;  tube  of  sterile  broth; 
aqueous-alcoholic  fuchsin. 

Method.  I.  For  Teeth.  1.  Place  a  small  drop  of  dis- 
tilled water  on  a  clean  cover-glass  or  slide. 

2.  Introduce  some  material  obtained  by  scraping  along 
the  base  of  and  between  the  teeth  with  a  sterile  platinum 
needle. 

3.  Allow  to  dry,  fix  and  stain  with  aqueous-alcoholic 
fuchsin. 

4.  Examine  microscopically  with  the  oil  immersion  lens. 

5.  Draw   all   forms   seen.     Would    all    of   these    forms 
grow  on  an  agar  plate?     Give  reasons  for  your  answer. 

II.  For  Throat  or  Nose.  1.  Prepare  a  swab  by  winding 
a  small  piece  of  absorbent  cotton  snugly  about  one  end  of 
the  wire  rod. 

2.  Place  in  a  test  tube,  swab   end  down,  and  prepare 
for  sterilization  as  with  pipettes. 

3.  Dry-sterilize. 

4.  Pour  an  agar  plate  and  allow  it  to  harden. 

5.  Moisten    the    sterile    swab    in    sterile    broth,    using 
aseptic  precautions,  and  then  swab  the  throat  or  nose. 

6.  Lightly  brush  the  inoculated  swab  over  the  surface 
of  the  agar  plate  and  place  the  plate  inverted,  at  37°,  to 
develop. 

7.  Using  the  same   swab,   make   a  smear  on   a   clean 
glass  slide,  dry,  fix,  stain  and  examine  as  with  the  prep- 
aration from  the  teeth. 

8.  Return  the  swab  to  the  tube  of  broth,  incubate  for 
twenty-four  hours  and  examine  the  growth  in  a  hanging 
drop. 


MICROFLORA  OF   THE  MUCOUS  MEMBRANE      151 

9.  Draw  and  describe  the  predominating  organisms. 

10.  Find  a  streptococcus,  if  possible,  on  the  agar  plate 
from  the  swab. 

11.  Make  a  stained  slide  and  have  the  instructor  in- 
spect the  same,  when  you  think  that  you  have  been  suc- 
cessful. 

12.  How  does  the  microflora  of  the  mucous  membrane 
differ  from  that  of  the  outer  skin? 

CAUTION.    Aseptic  precaution  must  be  taken  in  all  instances,  as 
some  of  the  microorganisms  may  be  pathogenic ! 

State  in  detail  your  results  from  I  and  II,  draw  any 
conclusions  possible  and  point  out  any  practical  applications. 

REFERENCES 

MARSHALL:  Microbiology,  pp.  522-524,  528,  546,  591-599,  609-613. 
BESSON:    Practical  Bacteriology,  Microbiology  and  Serum  Therapy, 
pp.  81,  191,  197,  270,  592-610,  617-626. 


PART   II 
PHYSIOLOGY  OF  MICROORGANISMS 


EXERCISE  1.     TO  DEMONSTRATE  THE  SMALL  AMOUNT 
OF   FOOD    NEEDED   BY   BACTERIA 

Apparatus.  Distilled  water;  eleven  sterile  Petri  dishes; 
sterile  1  c.c.  pipette;  sterile  10  c.c.  pipettes;  eight  sterile 
200  c.c.  flasks;  eleven  tubes  of  sterile  agar  (ordinary). 

Cultures.     B.  coli. 

Method.  1.  Place  150  c.c.  of  distilled  water  in  each  of 
two  sterile  flasks. 

2.  Sterilize  one  flask  (flask  B)  in  the  autoclav  for  ten 
minutes  at  15  Ibs.  pressure  (120°  C.). 

3.  Plate  1  c.c.  from  the  remaining  flask  (flask  A),  imme- 
diately on  agar. 

4.  As  soon  as  flask  B  is  cold,  plate  1  c.c. 

5.  Then  inoculate  the  water  in  flask  B  with  B.  coli,  using 
the  straight  needle  and  transferring  a  very  small  amount,  and 
plate  1  c.c. 

6.  Place  flasks  A  and  B  and  the  plates  made  from  the 
flasks  at  room  temperature. 

7.  Prepare  four  90  c.c.  and  two  99  c.c.  dilution  flasks  and 
sterilize. 

8.  At  the  end  of  five  days,  plate  from  flasks  A  and  B, 
using  1  c.c.  direct  and  dilutions  1  :  10,  1  :  100  and  1  :  1000. 
Incubate  the  plates  at  room  temperature. 

9.  Count  each  set  of  plates  at  the  end  of  five  days' 
incubation. 

152 


PHYSIOLOGICAL   CLASSIFICATIONS  153 

10.  Compute  the  weight  of  the  bacteria  in  the  flask  of 
distilled  water  at  its  highest  count. 

What  is  the  smallest  amount  that  may  be  weighed  on  the 
ordinary  analytical  balances?     Conclusions? 

11.  Plot  curves  to  show  whether  bacteria  are  decreasing 
or  increasing.     Offer  a  logical  explanation  for  the  direction 
the  curve  takes  in  each  instance. 

12.  Note  the  conditions  under  which  distilled  water  is 
obtained  and   dispensed  in  the  laboratory.     Why  is  the 
distilled  water  not  sterile? 

13.  By  what  process  of  distillation  may  distilled  water  be 
obtained  free  from  microorganisms?     What  several  factors 
outside    of   errors  in   technic   may   have  influenced  your 
results? 

14.  What  would  be  the  comparative  influence  of  a  large 
and  a  small  inoculation  upon  the  number  of  B.  coli  surviv- 
ing the  5  days  sojourn  in  the  distilled  water? 

16.  State  your  results  in  detail,  draw  any  possible  con- 
clusions and  point  out  any  practical  applications. 

REFERENCES 

MARSHALL,  C.  E.:   Microbiology,  pp.  88-89. 

FISCHER,  ALFRED  :  Structure  and  Functions  of  Bacteria,  pp.  52-54. 
PRESCOTT  and  WINSLOW:    Elements  of  Water  Bacteriology,  3d  Ed. 
pp.  151-153. 

SOME   PHYSIOLOGICAL   CLASSIFICATIONS    OF 
BACTERIA 

Bacteria  are  often  classified,  in  general  terms,  according 
to  their  functions,  into: 

Saprogenic,  or  putrefactive  bacteria; 

Zymogenic,  or  fermentative  bacteria; 

Pathogenic,  or  disease-producing  bacteria. 
According  to  their  food  requirements,  into : 

Prototrophic,  requiring  no  organic  food  (e.g.,  nitrifying 
bacteria)  j 


154  GENERAL  MICROBIOLOGY 

Metatrophic,    requiring    organic    food    (e.g.,    zymogenic 

bacteria,  saprophytes  and  facultative  parasites) ; 
Paratrophic,  requiring  living  food  (e.g.,  obligate  para- 
sites);   (A.  Fischer). 

According  to  special  food  preferred,  into: 
Acidophile:  acid  loving; 
Halophile:  salt  loving; 
Saccharophile:  sugar  loving; 
Saprophile:   loving  dead  organic  matter; 
Coprophile:  loving  barnyard  manure. 
According  to  their  oxygen  requirements,  into : 

Aerobic:  requiring  atmospheric  oxygen  for  growth; 
Anaerobic:  requiring  the  absence  of  atmospheric  oxygen; 
Partial    anaerobic:     requiring    an   intermediate    oxygen 

tolerance. 

According  to  the  necessity  of  one  kind  of  food  or  environ- 
ment, into: 
Obligate:  indicating  absolute  requirements,  e.g.,  obligate 

parasite,  obligate  anaerobe; 

Facultative:    indicating   a  variability   in   requirements; 
the    word    following    indicates    the    condition    under 
which  the  organism  may  live  but  does  not  prefer  for 
growth,  e.g.,  B.  coli  is  a  facultative  anaerobe. 
According  to  their  metabolic  products,  into : 
Chromogenic,  or  pigment-producing  bacteria; 
Photogenic,  or  light-producing  bacteria; 
Aerogenic,  or  gas-producing  bacteria; 
Thermogenic,  or  heat-producing  bacteria. 
Chromogenic  bacteria  are  classified  in  accordance  with 
the  nature  and  location  of  the  coloring  matter  which  they 
elaborate,  as 

Chromophorus  bacteria,  the  pigment  being  stored  in  the 
cell  protoplasm  of  the  organism  analogous  to  the  chlorophyll 
of  higher  plants,  e.g.,  green  bacteria  and  red  sulphur  bac- 
teria, purple  bacteria. 

Chromoparous  bacteria,  true  pigment  formers.    The  pig- 


ANAEROBIC   CULTURE   METHODS  155 

ment  is  set  free  as  a  useless  excretion,  may  be  excreted  as  a 
colored  body  or  as  a  colorless  substance  which  becomes 
oxidized  upon  exposure  to  the  air.  Individual  cells  are 
colorless  and  may  cease  to  form  pigment,  e.g.,  B.  prodigiosuSj 
B.  ruber,  B.  indicus. 

Parachrome  bacteria.    The  pigment  is  an  excretory  prod- 
uct but  is  retained  within  the  cell,  e.g.,  B.  violaceus.     (Bei- 
jerinck.) 
According  to  their  temperature  relations,  into: 

Pecilothermic  (poikilothermic)  bacteria:    adaptability  to 

temperature  of  environment; 
Stenothermic    bacteria:     a     very    narrow    temperature 

range  (strict  parasites); 

Eurythermic  bacteria:    a  very  wide  temperature  range 
(metatrophic  bacteria),  often  30°  between  maximum 
and  minimum  temperatures. 
According  to  their  optimum  temperature,  into: 

Cryophilic  (psychrophilic,  term 

used  chiefly  for  water  organ-  Min.        Opt.  Max. 

isms)  bacteria 0°  C.  15°  C.  30°  C. 

Mesophilic  bacteria  (includes 

pathogenic  bacteria) 15°  C.  37°  C.  45°  C. 

Thermophilic  bacteria 45°  C.  55°  C.  70°  C. 

ANAEROBIC   CULTURE  METHODS 

The  cultivation  of  strict  anaerobes  is  accompanied  by 
certain  technical  difficulties  arising  from  the  necessity  of 
removing  all  traces  of  oxygen  from  the  medium  and  from 
the  atmosphere  to  which  this  medium  is  exposed.  It  is, 
therefore,  necessary  to  employ  special  apparatus  or  special 
methods  for  their  cultivation. 

The  recent  investigations  of  Tarrozzi,  which  have  been 
confirmed  by  others,  seem  to  show  that  oxygen  does  not  exert 
any  direct  harmful  effect  on  anaerobic  organisms,  but  that 
the  presence  of  free  oxygen  prevents  the  medium  furnishing 


156  GENERAL  MICROBIOLOGY 

the  nutritive  substances  necessary  for  anaerobic  life.  Anae- 
robic organisms  can,  in  fact,  as  Tarrozzi  has  shown,  be  grown 
in  the  presence  of  the  oxygen  of  the  atmosphere  by  simply 
adding  pieces  of  animal  tissue  or  some  reducing  agent  to 
the  culture  medium. 

Several  principles  are  employed  as  a  basis  for  the  different 
methods  of  anaerobic  cultivations,  as  follows: 

I.  Exclusion  of  air  from  the  cultivation. 

II.  Exhaustion  of  air  from: 

1.  The  medium  by  boiling.     This  should  always  imme- 
diately precede  the  inoculation  of  the  medium  for  anaerobic 
cultivations. 

2.  The  vessel  containing  the  medium  by  means  of  an  air 
pump,  i.e.,  cultivation  in  vacuo. 

III.  Absorption  of  oxygen  from  the  air  in  contact  with  the 
cultivation,  i.e.,  cultivation  in  an  atmosphere  of  nitrogen, 
by  means  of: 

1.  Chemical  action  upon  a  readily  oxidizable  substance 
in   a  sealed   vessel   containing   the   cultures,   e.g.,   sodium 
hydroxide  upon  pyrogallic  acid. 

2.  Burning  a  filter  paper  saturated  with  alcohol  in  a 
sealed  vessel.     (Moore.)     If  the  paper  is  well  saturated  no 
deleterious  products  of  combustion  are  formed  which  would 
inhibit  growth. 

3.  Adding  to  the  medium  some  easily  oxidizable  sub- 
stance as  dextrose  (2%),  sodium  formate  (0.5%),  sodium 
sulphindigotate    (0.1%)    or  fragments  of  sterile   tissue   to 
absorb  all  the  available   oxygen  held  in  solution  by  the 
medium. 

The  chemicals  are  generally  employed  in  the  case  of  deep 
stab  cultures,  the  fragments  of  sterile  tissue  in  broth  cul- 
tures (Tarrozzi's  method).  The  tissue  must  be  freshly 
removed  from  an  animal  (rabbit,  mouse,  guinea  pig,  etc.) 
and  only  pieces  of  liver,  spleen,  kidney  or  lymphatic  glands 
may  be  used  with  success;  blood,  milk,  or  the  connective 
tissues  are  useless  for  the  purpose.  Vegetable  tissue  (potato, 


ANAEROBIC  CULTURE  METHODS  157 

elder  pith,  mushrooms,  etc.)  have  been  used  similarly  with 
success  (Wrzosek,  Ori  and  others).  Spongy  platinum  has 
also  been  used  similarly  with  satisfactory  results. 

The  vitality  of  anaerobic  organisms  is  exhausted  much 
more  quickly  on  media  prepared  on  these  principles  than  on 
media  under  anaerobic  conditions  (Jungano  and  Distaso). 

Perhaps  if  these  methods  were  used  in  conjunction  with 
anaerobic  methods  the  vitality  of  the  anaerobes  would  not 
be  impaired. 

4.  Growing  the  anaerobe  in  the  presence  of  a  vigorous 
aerobe  by  the  use  of  special  methods  or  apparatus. 

IV.  Displacement  of  air  by  an  indifferent  gas  such  as 
hydrogen,  carbon  dioxid,  etc. 

V.  A  combination  of  two  or  more  of  the  above  methods. 
The  following  methods  are  those  best  adapted  for  class 

use  and  can  be  utilized  in  a  regular  exercise  as  desired : 

I.     EXCLUSION    OF   AIR 

Hesse's  Method.  This  method  may  be  used  either  with 
a  pure  culture  or  for  determining  the  presence  of  anae- 
robes in  any  substance. 

Apparatus.  Tubes  of  agar  or  gelatin  for  stab  cultures; 
sterilized  oil  (olive  oil,  vaselin  or  paraffin  oil) ;  sterile  1  c.c. 
pipette. 

Culture.     Pure  culture  of  an  anaerobe. 

Method.  1.  Make  a  stab  culture  of  the  anaerobe,  using 
a  tube  containing  a  deep  column  of  the  medium,  and  thrust- 
ing the  inoculating  needle  to  the  bottom  of  the  tube.  The 
stab  culture  and  a  test  tube  shake  culture  also  may  be 
treated  as  follows: 

2.  With  the  sterile  pipette  place  a  layer  of  sterile  oil,* 
1  to  2  cm.  deep,  upon  the  surface  of  the  medium. 

3.  Incubate  at  the  optimum  temperature. 

*  Sterile  melted  agar  or  gelatin  may  be  substituted  for  the 
sterile  oil. 


158 


GENERAL  MICROBIOLOGY 


II.     EXHAUSTION    OF   AIR 

A.  By  Boiling.     It  is  well  to  expel  all  the  air  from  a 
medium  to  be  used  for  isolating  or  growing  anaerobes  by 
boiling  twenty  to  thirty  minutes,  and  cooling  rapidly  just 
previous  to  inoculating,  and  placing  under  anaerobic  con- 
ditions. 

B.  Cultivation  in  Vacuo.    This  requires  special  apparatus 
for  obtaining  a  vacuum  and  for  cultivation  in  some  cases. 


FIG.  39a. — Novy  Jar  for 
Tube  Cultures. 


FIG.  396.— Novy  Jar  for 
Plate  Cultures. 


Apparatus.     Special  tubes: 

1.  Vacuum  tubes  (Fig.  129,  p.  238,  Eyre's  Bacteriological 
Technic) . 

2.  Pasteur,  Joubert  and  Chamberland's  tube  (Fig.  80, 
p.  93,  Besson's  Practical  Bacteriology,  Microbiology  and 
Serum  Therapy). 

3.  Pasteur's  tube  (Fig.  81,  Besson,  ibid.). 

4.  Lacomme's  tube  (Fig.  82,  Besson,  ibid.). 

5.  Roux's  tube  for  stroke   cultures    (Fig.   91,   p.    101, 
Besson,  ibid.). 

6.  Roux's  tube  for  potato  cultures   (Fig.   92,   p.    101, 
Besson,  ibid.). 

7.  Esmarch's  tube  (Fig.  95,  p.  103,  Besson,  ibid.). 

8.  Vignal's  tube  (Fig.  96,  p.  103,  Besson,  ibid.). 


ANAEROBIC  CULTURE   METHODS 


159 


Special  flasks: 

1.  Pasteur's  flask  (Fig.  79,  p.  92,  Besson,  ibid.). 

2.  Flasks  with  long  necks  (Fig.  83,  p.  94,  Besson,  ibid.). 

3.  Bottle  (Fig.  84,  p.  94,  Besson,  ibid.). 

4.  Kitasato's  dish  (Fig.  93,  p.  10,  Besson,  ibid.). 

5.  Bombicci's  dish  (Fig.  94,  p.  102,  Besson,  ibid.). 

6.  Ruffer's  or  Woodhead's  flask  (Fig.  33,  p.  41,  Eyre, 
ibid.). 

Special  jars  in  which  test  tube  or  plate  cultures  may  be 
placed  and  a  vacuum  produced. 


FIG.  40. — Bulloch's  Anaerobic  Jar. 

1.  Novy's  jar  for  plates  (Fig.  135,  p.  245,  Eyre,  ibid.). 

2.  Novy's  jar  for  tubes  (Fig.  136,  p.  245,  Eyre,  ibid.). 

3.  Bullock's  anaerobic  apparatus  (Fig.  137,  p.  247,  Eyre, 
ibid.). 

4.  Tretrop's  apparatus  (Fig.  97,  p.  105,  Besson,  ibid.). 

5.  Botkin's  apparatus  (Fig.  134,  p.  244,  Eyre,  ibid.). 
Apparatus  for  obtaining  a  vacuum: 

1.  Electric  pump  adaptable  to  vacuum  or  pressure. 

2.  Water  vacuum  pump. 

3.  Mercury  vacuum  pump. 

Method.  1.  The  tube  and  flask  cultivations  are  prepared 
by,  (a)  placing  the  desired  medium  in  the  vessel ;  (6)  inoculat- 
ing from  the  desired  source;  (c)  attaching  to  the  vacuum 


160  GENERAL  MICROBIOLOGY 

pump  and  (d)  while  the  pump  is  running,  sealing  the  tube 
or  flask  in  the  flame,  at  the  constriction  provided  for  the 
purpose. 

2.  The  special  jars  have  the  advantage  that  tube  and 
plate  cultivations  may  be  prepared  in  the  usual  way  and 
then  placed  in  the  special  jar  which  is  then  attached  to  the 
vacuum  pump;  when  sufficient  vacuum  has  been  produced 
the  stopcock  is  turned  between  the  jar  and  the  pump. 

Isolation  of  anaerobic  organisms  may  be  accomplished 
with  much  greater  facility  by  the  use  of  these  jars. 

In  practically  every  instance  these  same  jars  may  also 
be  employed  in  the  methods  given  under  the  absorption  of 
oxygen. 

III.     ABSORPTION   OF   OXYGEN 

Different  methods  illustrating  this  general  principle  are 
much  used  because  of  its  simplicity  and  general  applicability. 
Any  vessel  with  a  tight  cover  as  a  Novy  jar,  an  ordinary 
chemical  desiccator,  a  Mason  fruit  jar,  etc.,  may  be  used 
as  a  container  for  the  tube  or  plate  culture. 

A.  Pyrogallic  add  method.  1.  Dry  pyrogallic  acid  is 
placed  on  top  of  some  absorbent  cotton  in  the  bottom  of 
the  jar  or  tube. 

2.  A  solution  of  sodium  hydroxide  is  poured  in,  but  not 
directly  upon  it. 

3.  The  cultures  are  put  in  place. 

4.  The  jar  or  tube  is  immediately  sealed  and   care  is 
taken  to  mix  the  chemicals.     The  organisms  thus  grow  in 
the  presence  of  the  inert  gas  nitrogen. 

The  chemicals  are  used  in  the  proportion  of  1  gm.  of 
pyrogallic  acid  to  10  c.c.  of  10%  aqueous  solution  of 
potassium  or  sodium  hydroxide  for  each  100  c.c.  of  air 
space. 

Apparatus.     Tubes  for  use  in  oxygen  absorption  method. 

1.  Simple  test-tube  method. 

2.  Giltner's  H  tube. 


ANAEROBIC  CULTURE  METHODS 


161 


3.  Buchner's  tube  (Fig.  130,  p.  239,  Eyre,  ibid.). 

4.  Turro's  tube  (Fig.  86,  p.  95,  Eyre,  ibid.). 

By  the  use  of  these  tubes  no  sealed  jar  is  necessary. 


FIG.  41. — Giltner's  Tube.     (Orig.)        FIG.  42. — Buchner's  Tube. 

1.  The  simple  test-tube  method  is  advantageous  in  that 
it  requires  no  special  apparatus.  It  has  disadvantages,  how- 
ever, which  will  be  mentioned  later. 

Apparatus.  Test  tube  of  sterile  medium;  rubber  stopper 
to  fit  tube;  pyrogallic  acid  and  sodium  hydroxide;  paraffin. 


162  GENERAL  MICROBIOLOGY 

Method.  1.  Inoculate  the  medium  with  the  material 
under  investigation  and  replace  the  plug. 

2.  Cut  off  the  plug  even  with  the  mouth  of  the  tube. 

3.  Push  the  plug  into  the  tube,  4  to  5  cm. 

4.  Place  on  top  of  the  plug  the  pyrogallic  acid  and  only 
enough  of  the  alkaline  solution  to  saturate  the  plug. 

5.  Insert  the  rubber  stopper  and  seal  with  paraffin  if 
necessary.     If  the  cotton  is  more  than  saturated,  the  strong 
alkaline  solution  will  run  through  the  plug  and  kill  the  organ- 
isms in  the  culture. 

This  preparation  is  valuable  only  for  noting  the  presence 
of  anaerobes  in  any  substance  or  studying  the  growth  of  an 
anaerobe  in  pure  culture,  on  account  of  the  difficulties  of 
technic. 

2.  Giltner's  H  Tube.     This  is  simply  two  test   tubes 
connected  near  their  mouths  by  a  short  piece  of  glass  tubing. 
By  this  method  the  tube  cultivation  may  be  placed  in  one 
test  tube,  the  chemicals  in  the  other  and  both'  tubes  stop- 
pered.    (Fig.  41,  p.  161). 

The  use  of  this  apparatus  presents  a  distinct  advantage 
over  any  other  tube  cultivation  method,  as  the  culture  is 
readily  discernible  at  all  times  and  may  be  handled  without 
the  disagreeable  features  of  the  other  methods. 

The  H  tube  lends  itself  also  to  the  method  depending 
upon  the  absorption  of  oxygen  by  an  aerobic  organism. 

3.  Buchner's  tube  consists  of  a  stout  glass  test  tube 
having  dimensions  of  about  23  cm.  in  length  and  4  cm.  in 
diameter,  fitted  with  a  rubber  stopper.  i   \ 

a.  A  test-tube  culture  of  the  organism  or  mixed  culture 
to  be  tested  is  prepared. 

6.  A    little    cotton,    the    pyrogallic    acid,    and    sodium 
hydroxide  solution  are  placed  in  the  Buchner  tube,  the  cul- 
ture immediately  introduced  and  the  rubber  stopper  imme- 
diately fitted  tightly  in  the  mouth  of  the  large  tube.     (Fig. 
42,  p.  161). 

4.  In  Turro's  tube,  the  medium  is  poured  through  the 


ANAEROBIC  CULTURE  METHODS  163 

small  inner  tube,  sterilized  and  inoculated.  The  pyrogallic 
acid  and  sodium  hydroxide  are  then  placed  in  the  bulb  and 
the  stopper  immediately  replaced. 

This  method  has  advantages  over  Buchner's  in  that  the 
oxygen  is  much  more  rapidly  absorbed  and  the  culture  is 
visible  during  incubation. 

Plates.     1.  Ordinary  deep  culture  (Petri)  dish. 

2.  McLeod's  plate  base  (used  with  the  bottom  of  a  deep 
Petri  dish).  (Muir  and  Ritchie,  6th  Ed.,  Fig.  23,  p.  66.) 

The  principle  of  using  these  two  plates  is  the  same 
throughout  and  is  illustrated  in  Exercise  2. 

Jars.  As  has  been  noted  before,  the  jars  designed  for 
obtaining  vacuum  may  be  utilized  in  the  pyrogallic  acid 
method  and  in  the  method  making  use  of  burning  alcohol 
to  exhaust  the  oxygen. 

B.  Liborius-Veillon  Method  and  Roux's  Biological 
Method  depend  upon  the  abstraction  of  oxygen  from  the 
medium  by  aerobic  organisms.  Liborius  makes  use  of  the 
aerobes  already  present  in  the  mixed  culture,  while  Roux 
uses  a  pure  culture  of  an  obligate  aerobe.  Nowak  first  grew 
Bad.  abortus  by  this  method. 

Liborius-Veillon  Method.  1.  Fill  long  test  tubes  (22 
cm.  XI. 5  cm.)  to  a  depth  of  10-15  cm.  with  glucose  agar  or 
gelatin  and  sterilize  (below  120°  C.). 

2.  Place  the  tubes  in  a  water  bath  and  boil  twenty  to 
thirty  minutes  to  liquefy  the  agar  and  drive  off  the  air  dis- 
solved in  the  medium;  then  cool  to  40°-45°  C.  until  sown. 

3.  Make  loop  dilutions  in  the  melted  agar  and,  as  soon  as 
the  tubes  are  sown,  cool  them  rapidly  in  an  upright  position. 

Aerobic  organisms  grown  in  the  upper  part  of  the  medium 
which  contains  a  certain  amount  of  air  in  solution,  while 
the  anaerobes  multiply  in  the  deeper  layer. 

Roux's  Method.  1.  Make  a  deep  agar  or  gelatin  stab  or 
shake  culture  of  the  organism  or  substance  to  be  studied. 

2.  Pour  upon  the  surface  of  this  medium  a  layer  1  to  2  cm. 
deep  of  a  broth  culture  of  a  vigorous  obligate  aerobe  as 


164  GENERAL  MICROBIOLOGY 

B.  subtilis,  or  an  equal  depth  of  liquefied  agar  or  gelatin  and 
inoculate  this  when  solid  with  the  aerobe. 

The  growth  of  the  aerobe  will  use  up  all  the  oxygen  that 
reaches  it  and  will  not  allow  any  to  pass  through  to  the 
medium  below,  which  will  consequently  remain  in  an 
anaerobic  condition. 

Giltner's  H-tube  Method.  The  use  of  Giltner's  H-tube 
allows  the  anaerobe  in  a  certain  medium  to  be  grown  on  one 
side  of  the  H  either  as  a  stab  culture  or  a  streak,  while  the 
aerobe  in  the  same  or  a  different  medium,  liquid  or  solid, 
may  be  grown  on  the  other  side.  Rubber  stoppers,  fitted 
to  mouths  of  both  tubes,  are  superimposed  on  cotton  plugs. 
The  aerobe  soon  exhausts  the  oxygen  from  the  tube,  allow- 
ing the  anaerobes  to  develop. 

This  is  the  method  recommended  for  determining  the 
presence  of  and  isolating  Bad.  abortus  from  infected  mucous 
membranes  and  tissues.  This  organism  when  first  isolated 
from  tissues  is  a  partial  anaerobe,  i.e.,  when  an  agar  shake 
culture  is  made  in  an  ordinary  test  tube  the  colonies  develop 
in  a  zone  about  0.5  cm.  in  width  about  1.5  to  2  cm.  below  the 
surface  of  the  agar. 

By  the  use  of  the  H  tube,  surface  colonies  of  this  organism 
may  be  readily  obtained  for  study. 

Novy  Jar  Method.  This  same  principle  may  be  applied 
by  the  use  of  separate  tube  or  plate  cultivations  of  anaerobes 
and  aerobes  in  a  Novy  jar  or  similar  apparatus;  the  aerobic 
organisms  should  be  offered  a  large  surface  for  growth  in 
each  case, 

IV.     DISPLACEMENT  OF  AIR  BY  INDIFFERENT  GASES 

The  special  tubes,  flasks  and  jars  adapted  to  cultivation 
of  anaerobes  in  a  vacuum  are  equally  applicable  in  this 
method. 

The  gas  generally  employed  is  hydrogen.  It  is  preferable 
to  other  gases  not  only  because  it  is  easily  prepared,  but  that 
it  has  no  injurious  effects  on  the  organisms. 


THE  EFFECT  OF  ANAEROBIC  CONDITIONS       165 

A  Kipp  generator  is  connected  up  with  three  wash  bottles, 
containing: 

(a)  10%  lead  acetate  solution  to  remove  H^S ; 

(6)  Silver  nitrate  solution  to  remove  AsHs ; 

(c)  10%  pyrogallic  acid  solution  made  alkaline  to  remove 
any  trace  of  oxygen,  may  be  used  to  furnish  hydrogen. 

Hydrogen  is  most  conveniently  obtained  by  keeping  a 
cylinder  of  the  compressed  gas  in  the  laboratory.  This 
gas  generally  contains  about  99.6%  hydrogen,  the  remain- 
ing 0.4%  is  mostly  or  entirely  air,  which  represents  0.08% 
oxygen.  The  gas  so  kept  requires  no  preliminary  washing, 
but  may  be  passed  direct  from  the  cylinder  into  the  jar 
or  flask. 

Carbon  dioxide  is  harmful  to  a  large  number  of  organ- 
isms, as  is  also  coal  gas.  Nitrogen  is  satisfactory,  but  its 
method  of  preparation  is  so  difficult  that  its  use  should  be 
abandoned  in  practical  bacteriology  unless  it  can  be  obtained 
compressed  in  cylinders. 

EXERCISE    2.     THE    EFFECT    OF    ANAEROBIC    CONDI- 
TIONS  UPON    MICROORGANISMS   FROM    MANURE 

Apparatus.  Modeling  clay;  tubes  of  sterile  gelatin; 
three  sterile  Petri  dishes;  three  sterile  deep-culture  dishes 
(use  top  of  Petri  dish  for  cover);  sterile  1  c.c.  pipettes; 
sterile  dilution  flasks;  six  tubes  of  sterile  agar;  pyrogallic 
acid;  10%  solution  of  sodium  hydrate;  absorbent  cotton. 

Culture.     Horse  manure. 

Method.  1.  Plate  the  manure  (1  gm.  in  99  c.c.  dilu- 
tion flask)  in  duplicate  in  the  Petri  dishes  and  in  the  deep 
culture  dishes,  using  dilutions  1  :  100,  1  :  10,000  and 
1  :  1,000,000. 

2.  As  soon  as  the  agar  is  solid,  invert  the  deep  culture 
dishes  containing  the  dilutions. 

3.  Place  a  small  piece  of  absorbent  cotton  in  the  center 
of  the  cover.     This  must  not  touch  the  agar. 

4.  On  the  absorbent  cotton,  place  1  gm.  of  pyrogallic 


166  GENERAL  MICROBIOLOGY 

acid  crystals;  then  place  10  c.c.  of  10%  NaOH  in  the  cover 
of  the  dish.  (The  cotton  prevents  a  too  rapid  reaction  be- 
tween the  chemicals.) 

5.  Seal  at  once  by  packing  the  space  between  the  cover 
and  bottom  air  tight  with  modeling  clay.     Then  mix  the 
chemicals. 

6.  Place  all  six  plates  at  room  temperature. 

Note.  In  the  reaction  which  takes  place  between  pyrogallic  acid 
and  NaOH,  oxygen  is  used  and  an  anaerobic  condition  is  established 
within  the  culture  dish  (exact  reaction  not  known.) 

7.  Count  the  organisms  after  seven  days.     Estimate  the 
number  of  different  types  of  colonies  developing  under  the 
varying  conditions  of  air  supply  and  note  growth.     Con- 
clusions? 

8.  Compare  your  results  with  those  of  others  and  draw 
conclusions. 

9.  Make  gelatin  stabs  of  three  or  four  of  the  predomi- 
nant   types    of    colonies    and    cultivate    anaefobically    by 
Hesse's   method.      What    types    of   organisms    are     these 
morphologically  and  culturally? 

10.  Are  any  types  found  on  aerobic  plates  which  are 
lacking  on  the  anaerobic  plates  and  vice  versa? 

What  type  of  anaerobe  is  frequently  found  in  horse 
manure? 

11.  When  do  anaerobic  conditions  exist  in  milk?     In 
soil?     Is  this  beneficial  or  otherwise  in  each  case?    What 
relation  may  there  be  between  age  of  milk  and  type  of  colo- 
nies?    Can  this    same  relationship  apply  in    the   case  of 
soil? 

12.  What  other  methods  may  be  used  for  obtaining 
anaerobic    conditions    for    microbial    growth?     Name    the 
obligate  anaerobes. 

13.  How  is  an  organism  isolated  which  is  tolerant  of  an 
amount  of  oxygen  less  than  that  of  the  atmosphere,  but  will 
not  grow  under  strictly  anaerobic  conditions? 

14.  State  your  results  for  the  experiment  in  detail  and 


ACIDS   FORMED    FROM  CARBOHYDRATES         167 

point  out  any  conclusions  that  may  be  drawn.     Mention 
any  practical  applications  to  be  made. 

REFERENCES 

EYRE:  Bacteriological  Technic,  2d  Ed.,  pp.  236-247. 
MARSHALL:  Microbiology,  pp.  93-98,  228-232,  306-331,  634-640. 
BESSON:    Practical  Bacteriology,  Microbiology  and  Serum  Therapy ,- 

pp.  87-105. 
GILTNER:   Suggestions  for  partial  anaerobic  cultures.     Science,  n.  s., 

Vol.  XLI,  No.  1061,  p.  663. 

EXERCISE   3.     TO   DEMONSTRATE   THAT   ACIDS   ARE 
FORMED   FROM    CARBOHYDRATES   BY  BACTERIA 

Apparatus.  Three  tubes  of  sterile  litmus  lactose  agar; 
three  tubes  of  sterile  dextrose  agar  containing  CaCO3; 
sterile  dilution  flask  (containing  about  150  c.c.  sterile  salt 
solution) ;  six  sterile  Petri  dishes-;  sterile  1  c.c.  pipettes. 

Culture.     Fresh  milk  culture  of  Bad.  lactis  addi. 

Method.  1.  Place  a  very  small  loopful  of  the  Bad.  lactis 
addi  culture  in  the  dilution  flask.  (Transfer  from  the  white 
portion  of  the  litmus  milk  culture.)  Shake  well.  . 

2.  Make  three  plates  from  each  medium,  using  widely 
varying  amounts,  for  example,  0.5  c.c.,  0.1  c.c.  and  1  drop. 
Just  before  plating,  mix  the  CaCOs  well  with  the  agar  (avoid 
air  bubbles). 

3.  Place  the  plates  (inverted)  at  room  temperature. 

4.  Examine  each  daily  after  forty-eight  hours.     Note 
how  each  medium  is  changed  by  the  growth  of  the  colonies. 
Explain  what  has  happened. 

How  is  the  object  of  the  experiment  demonstrated  in 
the  case  of  each  medium? 

6.  Compare  the  size  of  colonies  on  the  different  media; 
also  on  each  dilution  of  one  medium;  explain.  Why  are 
the  colonies  smaller  on  the  thickly  seeded  plates? 

6.  Write  the  chemical  equation  with  a  specific  enzyme 
for  each  change  in  the  case  of  each  medium. 


168  GENERAL  MICROBIOLOGY 

7.  Write  the  reaction  involving  the  CaCOs. 

8.  State  your  results  for  the  experiment  in  detail,  draw 
any  conclusions  and  point   out  any  practical  applications 
that  may  be  made. 


FIG.  43. — Solution  of  Calcium  Carbonate  by  Bact.  lactis  acidi. 
(Orig.  Northrup.) 

REFERENCES 

LOHNIS,  F.:    Laboratory  Methods  in  Agricultural  Bacteriology,  pp. 

71-73. 

VERNON:  Intracellular  Enzymes,  p.  98. 

EULER-POPE:  General  Chemistry  of  the  Enzymes,  pp.  31  and  58. 
MARSHALL:  Microbiology,  pp.  96,  102,  107,  108,  306-312. 
RAHN,  O. :  Tech.  Bui.  No.  10,  Mich.  Experiment  Station,  Fermenting 

Capacity  of  a  Single  Cell  of  Bact.  lactis  acidi,  pp.  22-23. 


ORGANIC  ACIDS   SERVE  AS  A  FOOD  169 

EXERCISE  4.     TO  SHOW  THAT  ORGANIC  ACIDS  MAY 
SERVE   AS   A  FOOD   FOR   SOME   ORGANISMS 

Apparatus.  Two  sterile  200  c.c.  Erlenmeyer  flasks; 
sterile  5  c.c.  pipettes;  200  c.c.  whey,  soured  by  Bact.  lactis 
acidi. 

Cultures.     Oospora  lactis. 

Mycoderma  (pickle  scum  yeast). 

Method.     1.  Titrate  the  acid  liquid  and  record  the  titre. 

2.  Place  100  c.c.  in  each  flask. 

3.  Heat  for  one  hour,  cool  and  inoculate  each  flask  with 
one  organism. 

4.  Titrate  every  two  days  from  the  time  growth  shows 
until  the  reaction  becomes   constant.     Always  use  sterile 
pipettes  for  obtaining  a  sample  for  titration. 

5.  Plot  curves  on  the  same  paper,  using  the  same  zero 
point. 

6.  Did  you  place  the  organism  in  its  natural  habitat? 
Will  either  of  these  organisms  use  another  acid  except 

that  common  to  its  habitat? 

7.  What  is  the  chemical  nature  of  this  organic  acid? 

8.  What  has  happened  to  the  organic  acid  in  question? 
Write  the  chemical  equation  showing  this  action. 

9.  What  type  of  enzyme  is  concerned  in  the  change  which 
takes  place? 

10.  State  your  results  in  detail  and  point  out  any  con- 
clusions to  be  drawn.     Point  out  any  practical  applications 
that  may  be  made. 

REFERENCES 

LAFAR:  Technical  Mycology,  English  Ed.,  Vol.  II,  Part  2,  pp.  417,  418, 

452-455. 
LASER:    Biological  Test  for  Butter.     Zeitschrift  fur  Hygiene  (1891), 

Vol.  X,  p.  513. 

MARSHALL:  Microbiology,  p.  111. 
NORTHRUP:    Tech.  Bui.  No.  15,  Michigan  Experiment  Station.     The 

Influence  of  Certain  Acid-destroying  Yeasts  upon  Lactic  Bacteria, 

pp.  5-7. 


170  GENERAL  MICROBIOLOGY 


EXERCISE  5.  TO  DEMONSTRATE  THE  VARIATION  IN 
FOOD  REQUIREMENTS  OF  BACTERIA  AND  THEIR 
SELECTIVE  POWER 

Apparatus.  Two  tubes  of  sterile  fermented  agar;  two 
sterile  Petri  dishes;  potassium  phosphate,  di-basic;  aspara- 
gin;  peptone;  ammonium  sulphate;  sodium  nitrate;  dex- 
trose; lactose;  saccharose. 

Culture.     B.  prodigiosus. 

Method.  1.  Melt  the  tubes  of  agar  in  the  steam  and, 
when  cool  but  still  liquid  (about  40°  C.),  inoculate  each 
heavily  with  B.  prodigiosus  and  pour  the  plates.  Allow  to 
stand  twenty-four  hours  at  room  temperature  before  pro- 
ceeding. Is  there  any  visible  growth  on  the  plate? 

2.  Mark  on  the  bottom  of  each  plate  with  drawing  ink, 
dividing  it  into  three  equal  sectors. 

3.  Use  ink  to  indicate  the  places  of  chemicals,  which 
should  be  deposited  at  the  center  of  each  plate  and  of  each 
sector. 

4.  Use  very  small  quantities  of  the  chemicals  and  be 
very  careful  not  to  scatter  them  over  the  plate  while  convey- 
ing them  to  their  proper  places,  otherwise  the  purpose  of  the 
experiment  will  be  defeated. 

5.  Incubate  at  room  temperature  and  examine  the  plate 
from  day  to  day  for  growth. 

Does  the  fermented  agar  support  growth  of  itself? 
What  explanation  can  you  give  for  the  action  which 
occurs? 

6.  How  is  the  variation  in  food  requirements  of  B.  pro- 
digiosus   shown?     The    selective   action?        Give    another 
example  of  the  demonstration  of  the  selective  action  of 
bacteria.     Which    source    of    nitrogen    is    seemingly    least 
available?     Which  most  available?     Why?     Which  carbo- 
hydrate is  most  easily  digested?     Which  least?     Why? 

Beijerinck,  knowing  that  agar  in  and  of  itself  is  a  food 
for  but  very  few  microorganisms,  reasoned  that  this  sub- 


SPLITTING  OF  CARBOHYDRATES  INTO  ALCOHOL    171 

stance  might  be  used  for  making  solid  synthetic  media 
if  it  could  be  freed  in  some  way  from  all  traces  of  food 
materials.  This  he  hoped  to  accomplish  by  allowing 
the  agar  to  ferment  spontaneously  upon  the  addition  of 
water. 

In  order  that  agar  may  not  support  microbial  growth 
it  must  be  allowed  to  ferment  over  a  long  period  of  time  to 
exhaust  every  possible  trace  of  food. 

7.  State  your  results  in  full,  draw  any  conclusions  that 
follow  and  point  out  the  practical  applications  that  may 
be  made. 

REFERENCES 

MARSHALL:  Microbiology,  pp.  89-93,  98-100. 

FISCHER,  A.:  Structure  and  Functions  of  Bacteria,  p.  115. 

EXERCISE  6.     TO  DEMONSTRATE  THE  SPLITTING  OF 
CARBOHYDRATES   INTO   ALCOHOL   AND    CO2 

Apparatus.  Clean  375  c.c.  Erlenmeyer  flask  fitted  with 
one-hole  rubber  stopper  containing  a  bent  glass  tube  plugged 
at  the  end  with  cotton;  two  calcium  chloride  tubes;  potash 
bulb ;  calcium  chloride  (small  granules) ;  potassium  hydrox- 
ide solution  (1  part  KOH,  2  parts  H20);  rubber  tubing  for 
connecting  up  apparatus;  400  c.c.  fractional  distillation 
flask;  thermometer;  250  c.c.  5%  saccharose  bouillon. 

Culture.     Sacch.  cerevisice. 

Method.  1.  Place  the  saccharose  broth  in  the  375  c.c. 
flask,  insert  the  rubber  stopper  and  sterilize  by  the  discon- 
tinuous method. 

2.  When  sterile,  inoculate  with  the  yeast  and  connect 
the  flask  in  "  train  "  with  a  CaCk  tube  (to  remove  moisture) 
a  tared  potash  bulb  (to  take  up  €62)  and  a  second  CaCl2 
tube. 

3.  Place  at  25°  to  30°  C.  and  allow  to  stand  until  no  more 
gas  evolves  (about  two  weeks) . 

4.  Test  quantitatively  for  alcohol  (distill  off  over  10  c.c. 


172  GENERAL  MICROBIOLOGY 

of  liquid,  measure  the  distillate  and  determine  its  specific 
gravity*)  and  estimate  percentage  of  yield. 

6.  Weigh  the  potash  bulb  to  find  the  amount  of  CO2 
given  off.  Does  it  correspond  to  the  yield  of  alcohol? 
Explain.  Are  your  results  according  to  theory? 

6.  Write  the  chemical  equation  for  each  change,  giving 
the  specific  enzymes  concerned  in  each  reaction.     What 
types  of  enzymes  are  concerned? 

7.  Would  alcohol  be  formed  in  bouillon  containing  no 
sugar?     In  a  5%  aqueous  solution  of  sugar?    Why? 

What  fermentable  substances  are  present  in  ordinary 
meat  bouillon? 

8.  State  your  results  in  full  and  draw  any  conclusions 
warranted.     What  practical  applications  may  be  made  of 
the  above? 

REFERENCES 

MARSHALL:  Microbiology,  pp.  135,  140-141. 

LAFAR:  Technical  Mycology,  Vol.  II,  Part  II,  pp.  473-481,  511-515. 

HAWK:  Physiological  Chemistry,  4th  Ed.,  pp.  255,  357. 


EXERCISE  7.  TO  DEMONSTRATE  THE  NECESSITY 
OF  NITROGEN  IN  SOME  FORM  FOR  MICROBIAL 
GROWTH 

Apparatus.     Four  tubes  each  of: 

Ordinary  broth  (organic  nitrogen,  soluble  albumins  and 
proteins) . 

Dunham's  solution  (organic  nitrogen,  soluble  peptone, 
no  albumen). 

Uschinsky's  asparagin  medium  (organic  nitrogen,  pro- 
tein-free) . 

Cohn's  solution  (inorganic  nitrogen  combined  with 
organic  acid). 

*  Table  for  determining  per  cent  of  alcohol  from  specific  gravity 
in  Sadtler's  Industrial  Organic  Chemistry  (1912),  pp.  579-584. 


NITROGEN  FOR  MICROBIAL  GROWTH  173 

Winogradski's  medium  for  nitrate  formation  (inorganic 
nitrogen  combined  with  an  inorganic  acid) . 

Winogradski's  medium  for  symbiotic  nitrogen-fixation 
(nitrogen-free) . 

Cultures.  B.  subtilis;  Ps.  radicicola;  Aspergillus  niger; 
Sacch.  ellipsoideus. 

Method.  1.  Inoculate  heavily  a  tube  of  each  medium 
with  Ps.  radicicola.  Proceed  likewise  with  the  three  remain- 
ing organisms. 

2.  Record  the  growth  at  the  end  of  five  days.     What 
conclusions  may  be  drawn? 

3.  Compare  the  formulae  of  the  different  media  given 
above.     Organic   nitrogen   is   present   in   the   radical   NH2, 
inorganic,  NH4. 

4.  What  is  the  explanation  for  the  growth  of  one  organ- 
ism and  not  another  on  a  certain  medium? 

Do  the  organisms  obtain  their  carbon  from  an  organic 
or  an  inorganic  compound  in  each  case?  Is  organic  or  in- 
organic nitrogen  the  most  available  in  each  case?  What 
is  the  value  of  such  a  medium?  Why  are  other  chemicals 
added  besides  the  main  nutrient? 

Why  is  distilled  water  used  in  all  these  media?  To 
what  does  the  term  "  auxanography  "  refer? 

5.  Give  your  results  in  detail  and  draw  any  conclusions 
warranted.     What  practical  applications  may  be  made  of 
the  above? 

REFERENCES 

MARSHALL:  Microbiology,  pp.  91-93,  99,  242,  362. 
LAFAR:  Technical  Mycology,  Vol.  I,  pp.  361,  468. 
LOHNIS:  Laboratory  Methods  in  Agricultural  Bacteriology,  p.  58. 


174  GENERAL  MICROBIOLOGY 


EXERCISE  8.     TO   DEMONSTRATE  THE  PRODUCTION 
OF   H2S   BY   BACTERIA 

Apparatus.  Three  tubes  ordinary  gelatin;  tube  ordinary 
agar;  sterile  Petri  dish;  lead  carbonate,  0.1  gm. 

Cultures.  B.  coli  communis;  B.  mycoides;  B.  mesen- 
tericus  vulgatus. 

Method.  1.  Make  stabs  of  all  organisms  in  gelatin  and 
place  these  at  a  temperature  not  exceeding  20°  C. 

2.  Melt  a  tube  of  agar  and  while  hot  add  0.1  gm.  of 
lead  carbonate  to  the  tube  and  mix  well  by  rolling  it  vigor- 
ously between  the  hands  (avoid  air  bubbles) . 

3.  Pour  into  the  sterile  Petri  dish  and  when  cold  make  a 
streak  (2.5  cm.  long  and  3  cm.  apart)  of  each  organism  on 
the  plate  in  the  order  named.     Invert  and  place  at  25°  C. 

4.  Examine  the  gelatin  stabs  from  day  to  day  for  lique- 
faction; examine  the  plate  culture  at  the  same  time.     Note 
the  action  on  lead  carbonate  (Beijerinck's  test). 

5.  Write  chemical  equations  for  the  action  of  sulphur- 
eted  hydrogen  on  lead  carbonate. 

/°\ 

Lead  carbonate  =  Pb<Q     >C  =  0 

xcr 

6.  Is  there  any  relation  between  the  power  of  organisms 
to  liquefy  gelatin  and  to  produce  "  lead-blackening  "  sul- 
phur?    Explain. 

From  what  compounds  is  the  H^S  produced  in  this 
experiment?  What  type  of  organisms  can  be  detected  by 
this  test?  Where  do  they  occur  in  the  largest  numbers  in 
nature? 

Which  of  the  ordinary  laboratory  media  offer  the  greatest 
source  from  which  this  gas  may  be  produced?  Explain. 

By  what  other  means  may  H2S  production  by  bacteria 
be  demonstrated? 


CHEMICAL  AGENCIES  ON  MICROBIAL  PIGMENT     175 

7.  Give  your  results  in  full.  Draw  any  conclusions 
possible  and  point  out  any  practical  applications  that  may 
be  made. 

REFERENCES 

LAFAB:  Technical  Mycology,  Vol.  II.,  Part  2,  pp.  558-560. 
MARSHALL:  Microbiology,  pp.  113-117. 

LOHNIS:  Laboratory  Methods  in  Agricultural  Bacteriology,  pp.  42,  116. 
EYRE;  Bacteriological  Technic,  2d  Ed.,  1913,  pp.  290-291. 

EXERCISE  9.  THE  EFFECT  OF  PHYSICAL  AND  CHEM- 
ICAL AGENCIES  ON  MICROBIAL  PIGMENT  AND 
THEIR  FORMATION 

Apparatus.  Six  tubes  of  gelatin;  six  dextrose  agar  slants 
+  15°;  eight  tubes  of  plain  milk,  sterile;  hydrochloric  acid; 
sodium  hydroxide;  chloroform;  ether;  benzol;  carbon 
bisulphide;  litmus  paper;  clean  slide;  small  funnel. 

Cultures.  Ps.  pyocyanea;  R.  violaceus;  B.  prodigiosus; 
Sarcina  lutea;  Torula  rosea;  B.  cyanogenus;  Bad.  lactis  acidi. 

Method.     A .  Effect  of  temperature  on  pigment  formation. 

1.  Make    two    dextrose   agar   streak    cultures  of    each 
organism. 

2.  Place  the  cultures  in  duplicate  at  25°  C.  and  37°  C. 

3.  Examine  every  day  or  so  and  at  the  end  of  a  week 
record  the  degree  of  pigment  formation  by  +  +,  +,  H — ,  — . 
Brightness  of  pigment  formation  should  be  considered  in  all 
cases,  not  the  amount  of  growth. 

4.  Where  is  the  pigment  seen  macroscopically  in  each 
case?     Explain. 

Does  temperature  have  any  influence  on  pigment  forma- 
tion? Does  this  correspond  in  each  case  with  that  of  the 
natural  habitat  of  the  organism? 

How  do  these  several  pigments  differ?  Of  what  impor- 
tance is  pigment  production? 

B.  Relation  of  air  to  pigment  formation. 

Make  gelatin  stabs  of  all  organisms  and  keep  at  or  below 
20°  C.  Note  the  place  of  pigment  formation.  Explain. 


176 


GENERAL  MICROBIOLOGY 


C.  Relation  of  light  to  pigment  formation. 

Make  two  streaks  of  B.  prodigiosus.  Place  one  in  bright 
sunlight,  keep  the  other  in  the  dark.  Explain  the  results. 

D.  Effect  of  chemicals  on  pigment. 

1.  To  one  of  the  brightest  pigmented  cultures  of  B. 
prodigiosus,  add  10  c.c.  of  95%  alcohol  and  shake  vigorously. 
Alcohol  dissolves  the  pigment. 

2.  Pour  off  into  a  flask  and  allow  to  settle.     Filter. 

3.  Divide  the  clear  filtrate  into  four  parts. 

To  one,  add  a  drop  or  two  of  HC1;  note  the  result  and 
explain.  To  the  second  add  a  drop  or  two  of  NaOH;  note 
the  result  and  explain. 

Place  the  third  in  bright  sunlight  and  note  what  happens. 

Place  a  few  drops  of  the  fourth  portion  on  a  clean  slide 
and  allow  to  evaporate  slowly.  Examine  crystals  under 
microscope  and  draw.  What  are  these  crystals?  Explain. 

E.  Solubility  of  pigment. 

1.  Make  five  dextrose  agar  streak  cultures  of  B.  pro- 
digiosus and,  when  well  pigmented,  try  the  solubility  of  the 
pigment  in  (a)  water,  (b)  chloroform,  (c)  ether,  (d)  benzol, 
(e)  carbon  bisulphide.  Results? 

•  2.  Are  any  of  the  different  bacterial  pigments  formed, 
water-soluble?  What  is  the  simplest  method  for  determin- 
ing whether  the  pigment  produced  by  an  organism  is  water- 
soluble? 

F.  Blue  milk  and  "  bloody  "  milk. 
1.  Inoculate  milk  tubes  as  follows: 


Organism. 

Alone. 

+  Bact.  lactia  acidi. 

ToTuld  rosea 

B    cyanogcnus 

B  prodigiosus. 

Control  

and    keep    at    25° 
Observe  daily. 


C.    along    with    uninoculated    control. 


PHYSICAL  PRODUCTS  OF  METABOLISM          177 

2.  At  the  end  of  seven  days  test  the  reaction  of  each.     Is 
there  any  relation  between  the  reaction  and  pigment  pro- 
duction? 

3.  What  conditions  are  conducive  to  the  formation  of 
red  milk?   of  blue  milk? 

How  would  you  describe  and  explain  "  bloody  "  milk  as 
produced  by  microorganisms  to  anyone  unfamiliar  with  the 
phenomenon?  How  differentiated  from  true  bloody  milk? 

4.  State  all  results  in  full.     Draw  any  conclusions  war- 
ranted and  point  out  the  practical  applications  that  may  be 
made. 

REFERENCES 

LAFAR:  Technical  Mycology,  Vol.  I,  pp.  105-122. 

THRESH:  Examination  of  Waters  and  Water  Supplies,  2d  Edition,  pp. 

43-44. 

STERNBERG:  Textbook  of  Bacteriology,  pp.  130-132. 
CONN:  Agricultural  Bacteriology,  pp.  156-157. 

EXERCISE  10.     TO  ILLUSTRATE  ONE  OF  THE  PHYSICAL 
PRODUCTS    OF   METABOLISM 

Apparatus.  Three  gelatin  slants  (20°  alkaline,  3%  salt) ; 
rubber  stopper  to  fit  one  of  the  gelatin  tubes. 

Culture.  Ps.  lucifera  or  some  actively  phosphorescing 
organism. 

Method.  1.  Make  a  streak  culture  of  the  above  organ- 
ism upon  each  of  the  gelatin  slants. 

2.  With  one  of  the  cultures,  boil  a  rubber  stopper  and 
insert  in  place  of  the  cotton  plug. 

3.  Place  the  stoppered  culture  and  a  second  one  (cotton- 
plugged)   at  20°  C.,  the  third  cotton-plugged  culture  at 
5°-10°  C. 

4.  Examine  in  the  light  and  in  the  dark  after  twenty-four 
and  forty-eight  hours.     Compare   (in  the  dark)   the  two 
cultures  at  20°  C.;  if  there  is  a  marked  difference,  loosen  the 
rubber  stopper  and  note  what  happens. 

5.  If  there  is  no  immediate  result  from  loosening  the 


178  GENERAL  MICROBIOLOGY 

stopper,  replace  the  stopper  with  a  sterile  cotton  plug  and 
note  both  cultures  after  twenty-four  hours.  What  occurs 
in  either  case? 

6.  Which  is  the  better  temperature  for  the  growth  of 
this  organism?     Can  you  suggest  a  reason  why?     What 
is  the  natural  habitat  of  this  type  of  organism?     Of  what 
importance  are  they? 

What  would  you  conclude  regarding  the  respiration  of 
phosphorescent  bacteria?  What  term  is  applied  to  bacteria 
exhibiting  this  phenomenon?  Of  what  importance  is  this 
phenomenon? 

7.  State  your  results  in  full,  and  draw  any  conclusions. 
What  practical  application  of  the  above  may  be  made? 

REFERENCES 

LAFAR:  Technical  Mycology,  Vol.  I,  pp.  123-126. 

MARSHALL:  Microbiology,  (1911),  p.  129 

FISCHER,  A.:  Structure  and  Functions  of  Bacteria  (1900),  pp.  63-64. 

ENZYMES:     CLASSIFICATIONS   AND    REACTIONS 

Enzymes  can  be  classified  in  several  different  ways : 

I.  According  to  their  place  of  activity  as  endo-enzymes 
(intracellular)  or  exo-enzymes  (extracellular) ; 

II.  According  to  the  type  of  food  substance  acted  upon, 
as    proteolytic    (protein-digesting),    lipolytic   (fat-digesting), 
enzymes  attacking  carbohydrates,  etc.; 

III.  The  most  satisfactory  and  inclusive  classification 
is    that    denoting    the    chemical    reactions    produced    by 
the  enzyme  during  its   activity.      Enzymes  may  thus  be 
called : 

1.  Hydrolytic,  the  addition  of  one  of  more  molecules  of 
water  to  the  molecule  of  the  substance  acted  upon. 

2.  Enzymes  producing  intramolecular  changes,  i.e.,  caus- 
ing a  rearrangement  of  the  atoms  within  the  molecule.     In  a 
few  cases  these  changes  may  be  hydrolytic  (urease)  but  a 


CLASSIFICATION  OF  ENZYMES  179 

number  of  the  enzymes  of  this  class  causes  this  rearrange- 
ment, splitting  the  molecule  without  the  addition  to  or  sub- 
traction from  any  elements  therein. 

3.  Oxidizing,  the  addition  of  oxygen  to  (or  the  subtrac- 
tion of  hydrogen  from)  the  molecule  of  the  substance  acted 
upon. 

4.  Reducing,   the  subtraction  of  oxygen  from   (or  the 
addition  of  hydrogen  to)   the  molecule  of  the  substance 
acted  upon.     The  reducing  enzymes  are  the  only  class  of 
enzymes  in  the  above  classification  acting  upon  inorganic 
compounds;   some  organic  compounds  are  also  acted  upon, 
viz.,  litmus,  methylen  blue,  etc. 

5.  Coagulating,    unknown    processes    accompanied    by 
coagulation.     Enzymes  whose  actions  are  not  so  well  known 
are  those  producing  syntheses,  isomers,  acting  anaerobically, 
etc. 

Note.  Euler's  suggestion  that  the  names  of  enzymes  be  formed  from 
the  compound  acted  upon,  by  suffixing  "-ase,"  will  be  adhered  to  in  all 
subsequent  study  of  enzymes,  the  suffix  u-lytic  "  for  the  adjective,  and  the 
suffix  "-ese  "  for  synthesizing  enzymes. 

Bayliss  has  suggested  the  ending  "-clastic"  for  the  adjective,  criticiz- 
ing the  ending  '-lytic"  because  the  definition  of  electrolytic,"  which 
must  be  granted  priority,  implies  action  by  the  agent  rather  than  upon 
the  substance  indicated  by  the  term.  He  also  questions  the  existence  of 
Euler's  "synthesizing  enzymes," 

CLASSIFICATION    OF    ENZYMES 

I.  Hydrolytic  Enzymes  of: 

A.  Carbohydrates,  including  Glucosides,  carbohydrases 

— general  term. 
1.  Polysaccharides.     (CeHioOs)^. 

a.  Celluloses:  cellulases — general  term. 
b'  Hemicelluloses :  cytases — general  term. 

c.  Starches,    insoluble    and    soluble:     amylases, 

(ptyalin,  diastase)— general  term. 

d.  Glycogens:  glycogenases — general  term. 

e.  Dextrins:  dextrinases — general  term. 


180  GENERAL  MICROBIOLOGY 


2.  Disaccharides. 

a.  Saccharose:    sucrase     (invertase,     invertin)— 

specific  term. 

b.  Lactose:  lactase  —  specific  term. 

c.  Maltose:  maltase  —  specific  term. 

3.  Glucosides:  glucosidases  —  general  term. 

a.  Amygdalin:  amygdalase   (emulsin,   synaptase) 

—  specific  term. 

b.  Tannin  (digallic  acid)  :  tannase  —  specific  term. 

4.  Pentoses:  (C5Hio05):r. 

a.  Pectoses:  pectases  —  general  term. 

B.  Esters:  ester  ases  —  general  term. 

1.  Fats:  Upases  (steapsin)  —  general  term. 
a.  Stearin:  stearinase  —  specific  term. 

C.  Proteins:  proteinases  or  carbamases  —  general  term. 
1.  Protein-digesting. 

a.  Proteins  broken  down  to  proteoses  and 
peptones  :  peptase  (pepsin  or  aci'd-proteinase) 

—  general  term. 

6.  Proteins  broken  down  further  to  polypeptids 
and  occasionally  to  a-amino  acids  with  a 
trace  of  ammonia:  tryptase  (trypsin  or 
alkali-pro  teinase)  —  general  term. 

c.  Proteoses,    peptones,    polypeptids    and    pro- 

tamins  broken  down  completely  to  a-arnino 
acids  with  a  trace  of  ammonia:  ereptase 
(erepsin,  protease)  —  general  term. 

II.  Enzymes  producing  intramolecular  changes,acting  on  : 
A.  Carbohydrates  (d-hexoses)  CeH^Oe  to  form: 

1.  Alcohol,    ethyl    and    carbon    dioxide:  zymase  — 

general  term. 

a.  Dextrose:  dextro-zymase  —  specific  term. 
6.  Levulose:  levulo-zymase—  specific  term. 
c.  Galactose:  galacto-zymase  —  specific  term. 

2.  Lactic  acid:    lactic  acid  bacteria  zymase  —  specific 

term. 


CLASSIFICATION  OF  ENZYMES  181 

B.  Acid   amides    (urea):    amidases — general   term,   to 

form : 
1.  Ammonium  carbonate:  urease — specific  term. 

III.  Oxidizing  enzymes:  oxidases — general  term,  of: 

A.  Ethyl  alcohol:    alcoholase  (alcoholoxidase,  vinegar- 

oxidase) — specific  term. 

B.  Organic  acids: 

1.  Lactic  acid:  lactacidase — specific  term. 

2.  Acetic  acid:  acetaddase— specific  term. 

C.  Tyrosin:  tyrosinase — specific  term. 

IV.  Reducing  enzymes:  reductases — general  term,  of: 

A.  Hydrogen  peroxide: 

1.  Catalase — specific  term,  free  oxygen  liberated. 

2.  Peroxidase — specific  term,  transference  of  oxygen. 

B.  Organic  dyes  to  leuco-compounds. 

1.  Methylen  blue,  litmus,   azolitmin,  indigo,  etc.: 

methylen-blue  reductase,  etc.— specific  term. 

2.  Methylen  blue  in  the  presence  of  formaldehyde 

(Schardinger's     reaction) :    perhydridase — spe- 
cific term. 

C.  Sulphur  to  £[28 :  sulphur  reductase — specific. term. 

D.  Nitrates  to  nitrites,  nitrates  to  NHs,  etc. :    nitrate-, 

nitrite-reductase,  etc. 

V.  Coagulating  enzymes. 

A .  Protein-coagulating. 

1.  Casein: 

a.  Of    cow's    milk:  caseinase     (rennin,    rennet, 

chymosin) — specific  term. 
6.  Of  human  milk:  parachymosin — specific  term. 

2.  Fibrin  of  blood:  thrombase   (thrombin) — specific 

term. 

B.  Carbohydrate-coagulating. 

1.  Pectin :  pectinase— specific  term. 


182  GENERAL  MICROBIOLOGY 

ENZYMIC  REACTIONS  OF  WELL  KNOWN  FERMEN- 
TATION  PROCESSES 

A.  Beer  or  bread  fermentation  by  Saach.  cerevisice. 

I.  starch      +  water +hydroly tic  enzyme  =  maltose. 

2(C6Hi0O6)  +  xH2O  +          amylase          =  zCi2H22Ou. 

II.  (a)      maltose    +  water  +hydrolytic  enzyme  =  2  mols.  dextrose. 

(from  yeast) 
Ci2H22On  +  H2O  +          maltase          =2C6H1206. 

III.  dextrose    +  enzyme  producing  intra-   =  alcohol   +   carbon 

molecular  change  dioxide. 

(from  yeast) 

C6H12O6    +  yeast  zymase          =  2CH3CH2OH+2CO2. 

This  same  yeast,  and  other  yeasts  also,  can  ferment  saccharose 
(.cane  sugar)  corresponding  to  II  above,  as  follows: 
II.  (6)    saccharose  -f- water -j-hydroly tic  enzyme  =  d-dextrose  + 
fj  (from  yeast)  d-levulose. 

Ci2H22On  +  H2O  +          sucrose  =C6H;2O6+C6Hi206. 

Both  of  these  simple  sugars  can  be  fermented  to  alcohol 
and  CO2  according  to  III  above. 

Comparatively  few  yeasts  can  attack  lactose  (milk  sugar) , 
e.g.,  Sacch.  kefir  (p.  369,  Marshall),  Sacch.  fragilis,  Sacch. 
tyricola,  etc.  (See  p.  106,  Guilliermond's  Les  Levures.) 
The  reactions  are  similar  to  II  (a)  above,  as  follows : 

(c)       lactose     +  water+hydrolytic  enzyme  =  d-dextrose +d-ga- 

(from  yeast)  lactose. 

Ci2H22On  +  H20  +  lactose  =  C6H12O6+C6Hi2O0 

Both  simple  sugars  are  changed  to  alcohol  and  CO2  according  to 
III  above. 

B.  Glucoside  decomposition,  by  molds,  bacteria  and  yeasts. 

General  reaction, 

glucoside  +  water  -f  hydroly  tic  enzy me  =  sugar  -f- aldehydes,  acids,  etc. 
Specific  reaction, 

amygdalin-f-  water -f  emulsin   =     dextrose      +  benzaldehyde  +  hy- 
drocyanic acid. 

C20H27OnN  +  2H2O  +  emulsin*  =     2C6H12O6      +  C6H6CHO+HCN. 
*  Emulsin  is  a  mixture  of  four  different  enzymes, 


ENZYMIC  REACTIONS  183 

C.  Fat  decomposition,  by  a  few  molds,  yeasts  and  bacteria. 
Only  microbial  method  of  fat  decomposition. 

General  reaction, 

f  at  +  water  +hydroly  tic  enzyme  =  fatty  acid  +  glycerin. 
Specific  reaction, 
stearin+3  mols.  water+steanrazse  =  3  mols.  stearic  acid+glycerin." 


H2O  C17H35COOH  CH2OH 
I  I 

C17H36CO-O-CH  +  H2O  =  Ci7H35COOH  +  CH2OH 
I  I 

Ci7H35CO.O-CH2       H20    CnH35COOH       CH2OH 

Lipases  decompose  more  especially  the  natural  fats, 
i.e.,  the  glycerin  esters  of  palmitic,  stearic  and  oleic  acids. 
Lipases  from  different  sources  differ  markedly  in  reactions. 

D.  Vinegar  fermentation. 

In  order  that  this  fermentation  may  take  place,  alcohol 
must  be  present  in  the  nutrient  solution,  either  added  arti- 
ficially or  as  a  product  of  fermentation.  In  the  latter  case, 
reactions  I,  Ha  and  III,  116  and  III,  or  He  and  III  under  A 
above  must  precede  those  of  the  vinegar  fermentation. 

Assuming  that  alcohol  is  present  in  the  liquid  in  which 
the  vinegar  bacteria  are  growing,  the  reactions  take  place  in 
two  stages,  as  follows:  (See  p.  448,  Marshall.) 

I.  ethyl  alcohol  -f  oxygen  +  oxidizing  enzyme  =  acetaldehyde+  water 
CH3CH2OH  +     O      +       alcoholase        =    CH3CHO     +  H2O. 

II.    acetaldehy  de+oxy  gen  +  oxidizing  enzyme  =  acetic  acid 
CHaCHO    +     O     +  acetaldehydase  =  CH3COOH 

If  there  is  not  plenty  of  air  present  the  oxidation  may  not 
become  complete  and  small  amounts  of  acetaldehyde  may 
form,  i.e.,  the  reaction  stops  at  the  first  stage. 

If  the  initial  percentage  of  alcohol  is  below  1  to  2%  the 
vinegar  bacteria  will  soon  attack  the  acetic  acid,  oxidizing 
it  completely  to  carbon  dioxide  and  water,  as  follows  : 

III.  acetic  acid  +oxy  gen  +  oxidizing  enzyme  =  carbon  dioxide+water 
CHsCOOH  +    4O     +       acetacidase       =         2CO2         +2H2O. 


184  GENERAL  MICROBIOLOGY 

This  cannot  take  place,  however,  if  above  10  to  12% 
acetic  acid  is  present,  as  this  amount  is  antiseptic  to  the 
vinegar  bacteria.  (See  pp.  450-451,  Marshall.) 

E.   Organic   acid   decomposition,  by  acidophile   organisms 
(organisms  of  the  Oidium  and  Mycoderma  type). 

lactic  acid  +oxygen +oxidizing  enzyme  =  carbon  dioxide  + water 
CH3CHOHCOOH  +  6O  +  lactacidase  =  3CO2  +3H2O. 

The  destruction  by  oxidation  of  acetic  acid  by  the  acetic 
bacteria  is  given  under  D  above. 

Nearly  all  organic  acids  are  decomposed  in  a  similar 
manner,  by  total  combustion. 

F.  Reactions  of  reductases. 

hydrogen  peroxide  —   oxygen   + reducing  enzyme  =  water +oxygen. 
H2O2  O        +         catalase         =  H2O  +      O. 

methylen  blue  +hydrogen  +    methylen   blue    =  leuco-basa  of 

reductase  methylen  blue. 

C6H3— N=(CH3)2  C6H3— N=(CH3)2 

NAs 


reductase  J 

C6H3=N=(CH3)2  C6H3—  N=(CH3)2 

G.  Lactic  acid  fermentation,  produced  in  milk  by 
Bact.  lactis  acidi. 

lactose  +  water  -fhydroly  tic  enzyme  =  d-dextrose-fd-galactose. 
Ci2H22Ou  +  H20  +  '         lactose  =      C6H12O6   +C6H12O6. 

dextrose    1  +enzyme    producing    intra-  =  lactic  acid. 
galactose  J  molecular  change 

c  acid  bacteria  zyraa*e  =  4CH3CHOHCOOH. 


Bact.  lactis  acidi  will  ferment  a  nutrient  solution  con- 
taining only  a  simple  sugar,  e.g.,  dextrose,  the  reaction  then 
being  according  to  the  second  equation. 


A  COMPARISON   OF  ACID  AND  RENNET  CURDS     185 

H.  Urea  fermentation,  by  urea  bacteria. 

Urea  +  water  +       hydrolytic  endo-enzyme       =  ammonium  carbonate. 
(producing  intramolecular  change) 


H2N 


\ 


C=0 


EXERCISE  11.     A  COMPARISON  OF  ACID  AND  RENNET 

CURDS 

Apparatus.  Three  200  c.c.  flasks  containing  100  c.c.  each 
of  sterile  skim  milk;  200  c.c.  fresh  skim  milk;  small  funnel; 
eighteen  large  test  tubes;  absorbent  cotton;  10%  lactic  acid; 
5%  phenol. 

Cultures.  B.  prodigiosus;  Bad.  lactis  addi;  B.  megater- 
ium. 

Method.  1.  Inoculate  flasks  containing  100  c.c.  sterile 
milk  with  B.  prodigiosus,  Bad.  ladis  addi  and  B.  megaterium. 

2.  Place  about  30  c.c.  fresh  skim  milk  in  a  200  c.c.  flask. 

3.  Add  10%  lactic  acid  drop  by  drop,  shaking  constantly. 

4.  When  the  first  finely  divided  curd  appears,  titrate. 
What  degree  and  percent  of  acid  were  necessary  to  curdle  the 
milk? 

5.  Titrate  fresh  skim  milk. 

6.  Prepare  150  c.c.  of  0.5%  phenol  milk  (by  adding 
15  c.c.  of  5%  phenol  to   135  c.c.  of  milk  and  sterilize). 
Mix  well  and  titrate  again.     Is  the  acidity  of  the  milk 
increased  perceptibly  by  the  addition  of  phenol? 

7.  As  soon  as  curd  appears  in  inoculated  flasks,  titrate. 
Determine  the  degree  and  percent  of  acidity  present. 

8.  Allow   the   cultures  to  develop   several    days    until 
decided  proteolysis   is   evident.     Then   titrate   again  and 


186  GENERAL  MICROBIOLOGY 

filter  each  culture  through  absorbent  cotton  (a  small  piece 
in  small  funnel);  15  c.c.  of  each  filtrate  is  necessary. 

9.  Mix  the  filtrate  from  each  culture  with  phenol  milk 
in  the  following  proportions : 

1 0.5  c.c.  filtrate +9. 5  c.c.  phenol  milk. 

2.  . 1.0  c.c.  filtrate+9.0  c.c.  phenol  milk. 

3 2.0  c.c.  filtrate+8.0  c.c.  phenol  milk. 

4 3.0  c.c.  filtrate+7.0  c.c.  phenol  milk. 

5 4.0  c.c.  filtrate+6.0  c.c.  phenol  milk. 

6 Heat  4  c.c.  of  filtrate  only,  in  steam 

for  fifteen  minutes.  After  cooling,  add  6  c.c.  phenol  milk. 
Shake  these  mixtures  well  and  incubate  at  37°  C. 

10.  Record  the  time  necessary  for  coagulation  in  each 
case.     Why  do  not  all  tubes  change  alike?     Explain. 

11.  Can  corrosive  sublimate  be  used  to  replace  phenol 
in  this  experiment?     Explain. 

What  types  of  enzymes  are  concerned  in  these  changes? 

Are  these  intra-  or  extra-cellular  in  each  case?  Will  the 
place  of  occurrence  of  the  enzymes  explain  the  action  taking 
place  in  the  different  sets  of  tubes? 

What  enzymes  produce  each  type  of  curd? 

What  are  the  differences  between  an  acid  and  a  rennet 
curd?  , 

Which  type  is  produced  by  each  of  the  organisms  used? 

What  effect  has  heat  upon  enzymes? 

12.  Give  results  in  full  and  draw  any  conclusions  per- 
mitted.    Point  out  any  practical  applications  of  the  above. 

REFERENCES 

LAFAR:  Technical  Mycology,  Vol.  I,  pp.  184-187. 

BAYLISS:  Nature  of  Enzymic  Action,  p.  37. 

EULER:  General  Chemistry  of  the  Enzymes,  pp.  45-48,  58. 

MARSHALL:  Microbiology,  pp.  139-141. 

VERNON:   Intracellular  Enzymes,  pp.  220-221. 

COHNHEIM:  Enzymes,  pp.  29,  87-89, 


PROTEOLYTIC  ENZYMES  UPON  GELATIN         187 

EXERCISE   12.     TO   SHOW  THE  ACTION   OF  PRO- 
TEOLYTIC  ENZYMES   UPON    GELATIN 

Apparatus.  Phenol,  0.5%  solution  (10  c.c.  of  5.0% 
phenol +90  c.c.  distilled  H2O) ;  water  bath  and  thermometer; 
gelatin,  7  gms.;  15  tubes  sterile  gelatin;  formalin;  xylol; 
5  sterile  1  c.c.  pipettes;  centimeter  scale. 

Cultures.  B.  ramosus;  B.  fluorescens;  B.  subtilis; 
B.  mycoides;  B.  prodigiosus. 

Method.  1.  Make  two  gelatin  stab  cultures  of  each 
organism  and  when  nearly  all  liquefied  (2-5  days  old)  pro- 
ceed with  the  experiment. 

2.  Dilute  the  5%  phenol  to  0.5%  as  above,  with  distilled 
water. 

3.  Add  7  gms.  gelatin,  dissolving  by  heating  not  over 
70°  C.     Neutralize  carefully. 

What  is  the  source  of  acid  in  phenol  gelatin?  Why  is 
the  gelatin  neutralized? 

4.  Select  five  test  tubes  having  the  same  diameter.     Fill 
each  half  full.     Solidify  in  an  upright  position. 

5.  Shake  each  of  the  liquefied  cultures  with  3  to  4  c.c. 
of  xylol. 

6.  After  one  hour,  add  1  c.c.  of  the  clear  supernatant 
xylol  solution  of  each  culture  to  a  tube  each  of  solid  phenol 
gelatin  and  of  ordinary  gelatin.     With  a  blue  pencil,  mark 
the  surface  of  the  solid  gelatin. 

7.  Examine  the  tubes  daily.     Is  there  any  evidence  of 
growth?     Of  liquefaction?     If  liquefaction  is  noted,  measure 
its  progress  in  millimeters. 

8.  Save    the    original    cultures    with    which    the    xylol 
has  been  shaken.     Is  there  any  evidence  of  further  growth? 

9.  If  1  c.c.  of  a  liquefied  gelatin  culture  of  a  liquefying 
organism  were  added  to  a  tube  of  solid  ordinary  gelatin, 
what  would  happen?     What  would  result  if  it  were  added 
to  a  tube  of  solid  phenol  gelatin?     Explain. 

10.  What  action  does  the  xylol  have?     How  can  you 


188  GENERAL  MICROBIOLOGY 

prove  that  xylol  has  this  action?     What  other  chemicals 
could  be  used  in  place  of  xylol? 

11.  What  is  the  object  of  adding  phenol  to  the  gelatin? 
Would  5%  phenol  serve  the  same  purpose?     Give  reason 
for  answer.     What  other  chemicals  could  be  used  in  place  of 
phenol?     Why?     What   chemicals   could    not   be   used   in 
place  of  phenol?     Why?     How  else  may  pure  enzyme  action 
be  demonstrated? 

12.  Add   5  drops  of  40%  formaldehyde   (formalin)   to 
each  tube  of  the  duplicate  liquefied  gelatin  cultures  and 
note  whether  they  become  solid  again  in  a. few  days.     Ex- 
plain the  action. 

13.  Give  your  results  in  full  and  draw  any  conclusions 
possible.     What  practical  applications  of  the  above  may  be 
made? 

REFERENCES 

BAYLISS:  Nature  of  Enzymic  Action,  p.  37. 

MARSHALL,  C.  E.:  Microbiology  (1911),  pp.  134,  138,  141. 

EULER,  HANS:  General  Chemistry  of  the  Enzymes  (1912),  pp.  115-123. 

VERNON,  H.  M.:  Intracellular  Enzymes  (1909),  pp.  215-220. 

EXERCISE   13.     TO   SHOW  THE  ACTION   OF   PRO- 
TEOLYTIC    ENZYMES    UPON   CASEIN 

Apparatus.  Five  tubes  of  sterile  milk;  two  tubes 
nutrient  agar;  sterile  5  c.c.  pipettes;  two  sterile  Petri  dishes. 

Cultures.  Bad.  lactis  acidi;  B.  ramosus;  B.  coli; 
B.  violaceus. 

Method.     1.  Warm  the  milk  (40-45°  C.). 

2.  Place  2  c.c.  in  each  sterile  Petri  dish  and  pour  one  tube 
of  melted  agar  upon  it,  mix  thoroughly  by  carefully  tilting. 

3.  When  solid,  make  parallel  streaks  with  Bact.  lactis 
acidi  and  B.  ramosus  upon  one  and  of  B.  coli  and  B.  viola- 
ceus upon  the  other.     Transfer  cultures -to  litmus  milk  also. 

4.  Examine  streak  cultures  every  day  for  evidences  of 
proteolysis.     Make  drawings  and  compare  the  rate  of  action 
of  the  different  bacteria.     Compare  streak  with  milk  cultures. 


ACTION  OF  ENZYMES  UPON  STARCH  189 

5.  Is  there  any  relation  between  the  power  of  enzymes 
to  liquefy  gelatin  and  their  ability  to  dissolve  casein?     What 
type  of  proteolytic  enzyme  dissolves  casein? 

6.  Give  your  results  in  detail.     Draw  any  conclusions 
which  follow  and  point  out  any  practical  applications  that 
may  be  made. 

REFERENCES 

MARSHALL,  C.  E. :  Microbiology,  pp.  138-139,  354. 

HASTINGS  :  The  action  of  various  classes  of  bacteria  on  casein  as  shown 

by  milk  agar  plates.     Cent.  f.  Bakt.,   II  Abt.,   Bd.   12   (1904) 

p.  590, 

EXERCISE  14.     TO  SHOW  THE  ACTION  OF  ENZYMES 
UPON    STARCH 

Apparatus.  Three  sterile  Petri  dishes;  three  test  tubes; 
soluble  starch;  three  tubes  sterile  agar;  Lugol's  iodin  solu- 
tion. 

Cultures.     Soil  for  inoculation. 

Method.  1.  Place  0.1  gm.  of  soluble  starch  in  each 
test  tube,  plug  and  sterilize  in  the  hot  air  sterilizer. 

2.  To  each  tube  of  starch  add  one  tube  of  melted  agar. 

3.  When  at  the  correct  temperature  (40°-45°  C.)  inoculate 
one   tube   with   one   loopful   of   soil.     (State   type   used.)  • 
Inoculate  the  second  from  the  first,  etc.,  then  plate  all  three 
dilutions. 

4.  When  the  colonies  are  well  developed,  pour  iodin  solu- 
tion on  the  plate  and  note  any  clearing  around  the  colonies. 
What  does  this  indicate? 

5.  Examine  microscopically  different  types  of  colonies 
attacking   starch.     Are   they   molds,   yeasts,    or   bacteria? 
Which  type  predominates  on  your  plates? 

6.  What  enzymes  are  concerned?     Give  specific  action. 
How  is  pure  enzymic  action  demonstrated? 

7.  Write  the   theoretical  chemical  equation.      What  is 
soluble  starch? 


190  GENERAL  MICROBIOLOGY 

8.  What  is  the  value  of  such  microbial  action  in  soil? 
Where    are    starch-digesting    microorganisms    present    in 
nature?     Of  what  importance? 

9.  State    results    in    full    and    draw    any    conclusions. 
Point  out  any  practical  applications  of  the  above. 

REFERENCES 

BAYLISS:  Nature  of  Enzymic  Action,  pp.  25,  113. 
MARSHALL,  C.  E.:   Microbiology  (1911),  pp.  90,  106,  248,  463,  etc. 
EULER,  HANS:  General  Chemistry  of  the  Enzymes,  pp.  13-15,  et  al. 
HAWK,  PHILIP  B.:    Physiological  Chemistry  (1914),  pp.   10,  48,  50, 

61,  65. 

SADTLER,  S.  P.:  Industrial  Organic  Chemistry,  p.  186. 
LAFAR:  Technical  Mycology,  Vol.  II,  Part  2,  pp.  351-353. 

EXERCISE  15.     TO  SHOW  THE  ACTION  OF  REDUCING 
ENZYMES 

Apparatus.  Petri  dish;  medium  fine  sand;  sulphur; 
cake  of  Fleischmann's  compressed  yeast,  fresh  (obtain 
this  yourself);  small  mortar  and  pestle;  lead  acetate 
paper. 

Method.  1.  Thoroughly  grind  the  sulphur,  sand  and 
yeast  cake  in  a  small  mortar. 

2.  Place  the  contents  of  the  mortar  in  a  covered  dish 
with  a  piece  of  moistened  lead  acetate  paper.     What  odors 
are  noted? 

3.  What  reaction  is  demonstrated  by  the  lead  acetate 
paper?     What  reactions  are  taking  place?     Give  a  chemical 
equation  which  will   cover  the  final  changes.     May  other 
enzymes  be  released  from  the  yeast  cells  during  the  process 
of  maceration?     If  so,  what  enzymes? 

What  names  are  applied  to  the  specific  enzyme  acting  on 
sulphur  and  to  the  class  to  which  it  belongs?  Where  does 
this  action  occur  in  nature? 

This  enzymic  action  was  first  observed  in  1888  by  a  Frenchman, 
J.  de  Reypailhade,  who  found  that  the  alcoholic  extract  of  yeast  would 
convert  elementary  sulphur  into  sulphuretted  hydrogen. 


ACTION  OF  THE  ENZYME  CATALASE  191 

4.  Give  all  results  in  full  and  draw  any  conclusions 
permissible.  What  practical  applications  may  be  made  of 
the  above? 

REFERENCES 

MARSHALL,  C.  E.:  Microbiology,  pp.  135,  142-143. 

LAFAR,  F.:  Technical  Mycology,  Vol.  II,  Part  II,  pp.  558-560. 

KRUSE,  W. :  Allegemeine  Mikrobiologie,  pp.  652-655. 

EXERCISE   16.     TO   SHOW  THE  ACTION   OF  THE 
ENZYME   CATALASE 

Apparatus.  Four  fermentation  tubes  of  nutrient  broth 
(sterile) ;  hydrogen  peroxide  (full  strength) . 

Cultures.  B.  coli;  B.  subtilis;  B.  mycoides;  Bad. 
lactis  acidi. 

Method.     1.  Inoculate  the  fermentation  tubes. 

2.  After  growth  is  well  started,  add  1  c.c.  of  hydrogen 
peroxide  to  each  tube  and  mix  well. 

3.  After  the  tubes  have  stood  for  half  an  hour  measure 
the  gas  formed.     Compare  your  results  with  those  of  other 
students. 

Note.  If  the  bottle  of  H2O2  stands  uncorked  or  in  a  warm  place 
it  decomposes  very  rapidly  and  the  gas  formed  in  the  fermentation 
tubes  will  be  much  less  than  from  a  full  strength  solution. 

4.  What    is    the     strength    of    commercial     hydrogen 
peroxide? 

Where  else  is  catalase  found?  What  is  the  type  of  action 
supposedly  taking  place?  Write  chemical  equation  showing 
the  general  reaction. 

Have  you  ever  observed  the  action  of  catalase  produced 
in  animal  tissues?  What  is  the  difference  between  catalase 
and  peroxidase? 

5.  State  the  results  of  your  experiment  in  full  and  draw 
any  conclusions  permissible.  Point  out  any  practical 
applications  that  may  be  made. 


192  GENERAL  MICROBIOLOGY 

REFERENCES 

BAYLISS:   Nature  of  Enzymic  Action,  pp.  140-141. 

EULER-POPE:  General  Chemistry  of  the  Enzymes,  pp.  65,  67-68. 

VERNON:  Intracellular  Enzymes,  pp.  127-132. 

LOHNIS,  F.:    Laboratory  Methods  in  Agricultural  Bacteriology,  pp, 

66-67,  79. 
MARSHALL;  Microbiology,  pp.  135,  142-143. 

EXERCISE    17.     TO    DEMONSTRATE    THE    OXIDIZING 
ENZYME    OF   VINEGAR   BACTERIA 

Apparatus.  200  c.c.  fermented  cider  (or  other  fruit 
juice);  sterile  375  c.c.  Erlenmeyer  flask;  sterile  10  c.c. 
pipette;  water  bath;  specific  gravity  bottle. 

Culture.     Bad.  aceti. 

Method.  1.  Place  the  cider  in  a  sterile  flask  and  heat 
in  a  water  bath  at  60°  C.  for  one  hour.  Cool  quickly.  What 
is  this  process  called? 

2.  Determine    the    specific    gravity  of    the    fermented 
cider. 

3.  Inoculate  with  a  pure  culture  of  Bact.  aceti  and  titrate 
every  three  days  until  the  titre  is  constant. 

4.  Plot  the   curve  showing  and  explain  the  direction 
which  the  curve  takes.     What  is  taking  place?     Enzyme? 
Chemical  equation? 

5.  Determine  the  specific  gravity  of  the  solution  at  the 
last  titration.  ..How  does  this  compare  with  specific  gravity 
of  cider  vinegar  of  legal  standard?     What  is  the  legal  stand- 
ard for  vinegar  in  this  state?     Can  you  explain  why  all 
vinegar  does  not  come  up  to  the  legal  standard? 

6.  Is  it  practicable  to  use  pure  cultures  for  preparing 
vinegar?     How  do  various  species  of  vinegar  bacteria  differ 
from  one  another? 

Under  what  conditions  will  acetic  fermentation  set  in 
"  spontaneously?  " 

What  raw  materials  will  give  rise  to  a  vinegar  by  a 
normal  acetic  fermentation? 


ACTIVATOR  FOR  ENZYMIC  ACTION  OF  RENNET     193 

How  does  a  scarcity  of  alcohol  influence  the  amount  of 
acid  produced?  An  excess  of  alcohol? 

How  may  vinegar  be  prepared  artificially?  How  adul- 
terated? 

7.  State  the  results  of  your  experiment  in  detail  and 
draw  conclusions.  Point  out  any  practical  applications 
that  may  be  made. 

REFERENCES 

EULER-POPE:  General  Chemistry  of  the  Enzymes,  60-61. 
MARSHALL:  Microbiology,  pp.  135,  142,  448-451. 
SADTLER:  Industrial  Organic  Chemistry,  pp.  266-272. 
LAFAR:  Technical  Mycology,  Vol.  I,  pp.  295-307. 
Circular  on  "  Vinegar  "  prepared  by  Bacteriological  Laboratory,  East 
Lansing,  Mich. 


EXERCISE  18.  TO  DEMONSTRATE  THE  NECESSITY 
OF  AN  ACTIVATOR  FOR  THE  ENZYMIC  ACTION  OF 
RENNET  (FROM  CALF'S  STOMACH) 

Apparatus.  Four  clean  200  c.c.  Erlenmeyer  flasks; 
sweet  skim  milk  (not  over  -f!5°);  rennet,  fresh  com- 
mercial; two  1  c.c.  pipettes;  sterile  saturated  solution  of 
monobasic  calcium  phosphate  (Ca(H2PO4)2+H2O);  water 
bath;  thermometer. 

Method.  1.  Place  about  150  c.c.  of  skim  milk  in  each 
of  two  200  c.c.  flasks,  plug  these  and  sterilize  them  by  the 
Tyndall  method. 

2.  When  ready  to  start  the  experiment,  obtain  300  c.c. 
of  fresh  skim  milk  and  place  150  c.c.  of  milk  in  each  of  the 
two  remaining  flasks. 

3.  Mark  the  fresh  milk  flasks  Nos.  1  and  2;  the  sterilized 
milk  flasks  Nos.  3  and  4. 

4.  Place    all    four    flasks    in    a    water  bath    and    heat 
the  water   to  35°   C.,   not  higher.     (Steam   cannot   be  sub- 
stituted.) 

5.  Mark  the  flasks  as  follows : 


194  GENERAL  MICROBIOLOGY 

Flask  No.  l=unheated  milk + rennet. 

11     2  =  unheated  milk + calcium  phosphate  -f  ren- 
net. 

"        ll     3  =  heated  (sterilized)  milk + rennet. 
"        "     4  =  heated  (sterilized)  milk + rennet + calcium 

phosphate. 

6..  Add  1  c.c.  of  the  calcium  phosphate  solution  to  one 
flask  of  fresh  milk  and  mix.  (Flask  No.  2). 

7.  To  each  flask  of  milk  add  a  drop  of  rennet,  shake 
quickly,  replace  the  flask  in  the  water  bath  and  leave  for  ten 
to  twenty  minutes  without  disturbing. 

8.  Add   1   c.c.  of  calcium  phosphate  solution  to  flask 
No.  4.     Shake  quickly,  return  the  flask  to  the  water  bath 
and  leave  for  ten  to  twenty  minutes  without  disturbing. 
Observe.     If  no  curd  appears,  set  the  flasks  at  37°  C.  and 
observe  after  about  twenty-four  hours.     What  is  the  expla- 
nation for  the  phenomena  occurring  in  this  flask? 

9.  Observe  the  milk  in  all  flasks  for  curdling.     Which 
flasks  of  milk  curdled?     Why? 

10.  What  are  the  various  synonyms  of  "rennet?" 
What  is  the  specific  action  of  this  enzyme? 

What  is  the  source  of  the  enzyme  used?     How  prepared? 

What  living  organisms  produce  coagulating  enzymes? 

Does  the  rennet  produced  by  various  bacteria  require 
soluble  calcium  salts  for  an  activator?  How  would  you 
determine  this? 

What  is  an  activator?  To  what  property  of  an  activator 
is  its  action  attributed?  What  are  the  different  classes  of 
activators?  Do  all  enzymes  require  activators? 

11.  Give  all  results  in  full  and  draw  any  conclusions  pos- 
sible.   What  practical  applications  of  the  above  may  be  made? 

REFERENCES 

LAFAR:  Technical  Mycology,  Vol.  I,  pp.  185,  186. 

BAYLISS:  Nature  of  Enzymic  Action,  pp.  120-121,  132-133. 

RICHMOND:  Dairy  Chemistry,  p.  301. 

EULER-POPE:  General  Chemistry  of  the  Enzymes,  pp.  94,  106-109. 


EFFECT  OF  CONCENTRATED  SOLUTIONS         195 


EXERCISE    19.     EFFECT   OF   CONCENTRATED 
SOLUTIONS   UPON   MICROORGANISMS 

Apparatus.  750  c.c.  nutrient  broth;  gelatin;  salt; 
dextrose;  saccharose;  five  10  c.c.  pipettes;  100  c.c.  graduate. 

Cultures.  Mycoderma;  B.  coli;  M.  varians;  Sacch. 
cerevisice;  Penicillium;  B.  prodigiosus. 

Method.  1.  Make  up  four  tubes  each  of  the  following 
concentrations : 

Electrolytes:  sodium  chloride,  5%,  10%,  15%,  20%,  25%. 

Non-electrolytes:  dextrose  and  saccharose,  30%,  45%, 
60%,  75%,  respectively. 

Colloids:  gelatin,  5%,  10%,  30%,  50%. 

2.  With  the  exception  of  the  gelatin  the  separate  weigh- 
ing out  for  each  concentration  can  be  avoided  by  using  the 
following  method  of  mixing,  with  the  stock  solution  con- 
taining 50%  or  75%  of  the  substance  under  study: 

(a)  Weigh  out  the  correct  quantity  of  material  and  place 
it  in  a  100  cc.  graduate. 

(6)  Fill  the  graduate  to  the  100  c.c.  mark  with  nutrient 
broth.  Place  the  hand  over  the  mouth  of  the  graduate  and 
shake  until  solution  is  complete.  If  necessary,  fill  to  the 
mark  again  with  broth.  For  example:  Dissolve  25  g.  of 
salt  in  about  90  c.c.  of  broth,  fill  the  graduate  to  100  c.c., 
to  obtain  a  broth  of  which  100  c.c.  contain  25  g.  of  salt. 
Mix  this  salt  broth  with  common  broth  in  the  following 
proportions,  by  means  of  pipettes: 

Salt  Plain  Salt  content 

broth.  broth.  of  mixture. 

2  c.c.  +  8  c.c.  5% 

4  c.c.  +  6  c.c.  10% 

6  c.c.  +  4  c.c.  15% 

8  c.c.  +  2  c.c.  20% 

10  c.c.  +  0  c.c.  25% 

Broth  will  give  a  precipitate  after  heating  with  salt, 
consequently  each  salt  broth  mixture  has  to  be  filtered 
separately  after  heating.  What  is  this  precipitated  material? 


196  GENERAL  MICROBIOLOGY 

Note  1.  The  stock  solution  of  dextrose  is  best  prepared  by  adding 
to  75  g.  dextrose,  50  c.c.  of  broth,  heating  the  mixture  in  the  steam 
until  dissolved  and  then  making  up  to  100  c.c.  with  broth.  The  sugar 
solutions  may  have  to  be  filtered  also. 

Note  2.  As  it  is  a  difficult  procedure  to  make  up  as  high  concen- 
trations of  gelatin  as  30%  and  50%  with  any  degree  of  ease  and 
accuracy,  gelatin  prepared  according  to  the  following  procedure  will 
serve  to  illustrate  the  point  of  the  exercise. 

With  a  blue  pencil,  mark  the  10  c.c.  level  on  each  of  sixteen  tubes. 
To  make  up  5%  gelatin,  place  0.5  gm.  gelatin  in  each  of  four  tubes 
and  make  up  to  the  10  c.c.  mark  with  broth.  Proceed  similarly  with 
the  remaining  concentrations.  After  heating  once,  mix  well  with  a 
sterile  platinum  loop. 

Gelatin  is  practically  the  only  colloid  that  can  be  obtained  in  solu- 
tions concentrated  enough  for  this  experiment  (up  to  70%).  Great 
care  must  be  taken  to  avoid  the  condensation  of  moisture  on  the  sides 
of  the  test  tubes  or  flask,  because  this  moisture  will  reduce  the  con- 
centration of  the  surface  gelatin  and  thus  cause  incorrect  data. 

3.  Sterilize  the  tubes  by  the  intermittent  method. 

4.  Inoculate  heavily  one  tube  of  each  concentration  of 
the  salt  with  Mycoderma,  one  with  B.  coli  and  one  with  M. 
varians.     Inoculate  tubes  of  each  concentration  of  dextrose, 
saccharose  and  gelatin  with  Penicilliwn,  Sacch.  cerevisice, 
and  B.  prodigiosus,  leaving  a  tube  of  each   concentration 
uninoculated  for  control. 

Note.  The  inoculation  must  be  heavy,  because  experience  teaches 
that  a  small  inoculum  is  sometimes  not  sufficient  to  secure  growth. 

5.  Note  and  tabulate  the  growth  after  seven  days. 

6.  Reinoculate    the   lowest    concentration    of    each    set 
that  does  not  show  growth  from   the  highest  of  the  same 
set    that    does   grow,   e.g.,    if   Penicillium   grows    at  45% 
dextrose  but  not  at  60%,  inoculate  60%  from  45%.     If 
it  now  grows,  what  is  indicated?     Is  there  not  plenty  of 
water  and  food  material  present?         Explain  your  results. 

Does  the  natural  habitat  or  food  requirements  of  each 
organism  explain  in  any. way  the  action  occurring? 

7.  What  is  meant  by  osmotic  pressure?     Electrolyte? 
Colloid?     What  is  known  of  the  relative  osmotic  pressure 


EFFECT  OF  DESICCATION  UPON  BACTERIA      197 

of  electrolytes,  non- electrolytes  and  colloids?  How  are 
these  differences  explained?  Is  the  relative  preserving 
power  of  these  different  substances  according  to  the  molec- 
ular weight  theory?  Explain. 

Why  is  a  large  inoculum  more  apt  to  insure  growth  than 
a  small  inoculum? 

Note.  Salt-resisting  organisms  can  be  secured  by  plating  in  agar 
containing  10  to  l7>%  of  salt,  from  butter,  brine  pickles,  salt  pork, 
salt  fish,  and  other  salted  food.  Sugar-resisting  organisms  can  be 
obtained  similarly. 

8.  State  the  results  of  your  experiment  in  full  and  draw 
conclusions.  Point  out  any  practical  applications  that  may 
be  made. 

REFERENCES 

MARSHALL:  Microbiology,  pp.  147-151. 

FISCHER,  A.:  Structure  and  Functions  of  Bacteria,  pp.  5,  8-9. 

EXERCISE  20.     THE  EFFECT  OF  DESICCATION  UPON 
BACTERIA 

Apparatus.  Four  sterile  cover-giasses ;  four  sterile 
Esmarch  dishes;  potato  knife;  eighteen  tubes  of  sterile  broth. 

Cultures.  B.  violaceus  (non-spore-producing,  non-slime- 
forming) ;  slimy  milk  bacillus  (non-spore-producing,  slime- 
forming);  B.  subtilis  (spore-producing,  non-slime-forming); 
meat  bacillus  (spore-producing,  slime-forming). 

Method.  1.  Using  a  platinum  needle,  smear  one  cover- 
glass  thickly  with  a  culture  of  B.  violaceus,  the  second  with 
the  slimy  milk  bacillus,  the  third  with  the  spore-former 
(B.  subtilis)  and  the  fourth  with  the  spore  and  slime  produc- 
ing meat  bacillus. 

2.  Place  each  of  these  cover-glasses  in  separate  sterile 
Esmarch  dishes  and  break  each  into  five  or  six  small  pieces 
with  a  sterile  potato  knife. 

3.  Transfer  a  piece  of  each  cover-glass  to  a  tube  of 
nutrient  broth  after  1,  3,  7,  14,  etc.,  days. 


198  GENERAL  MICROBIOLOGY 

Stop  transferring  when  you  find  that  there  is  no  growth 
in  the  test  tube  last  inoculated. 

4.  What  influence  has  the  physical  condition  of  the  sub- 
strate upon  which  the  microorganisms  are  dried  upon  their 
longevity?     Illustrate. 

What  dried  cultures  of  microorganisms  have  been  used 
with  success  commercially?  Without  success?  What  other 
methods  may  be  employed  to  demonstrate  the  effect  of 
desiccation  on  microorganisms? 

5.  State  all  results  in  full  and  draw  any  conclusions. 
Point  out  any  possible  practical  applications. 

REFERENCES 

MARSHALL:    Microbiology,  pp.  151-152,  280-283,  338-343,  367-369, 

374-380,  428-429,  453-460. 

SMITH,  E.  F.:  Bacteria  in  Relation  to  Plant  Diseases,  Vol.  I,  pp.  70-71. 
EYRE:  Bacteriological  Technic,  2d  Ed.,  pp.  306-308. 

EXERCISE  21.  THE  DETERMINATION  OF  THE  OPTI- 
MUM, MAXIMUM  AND  MINIMUM  TEMPERATURE 
REQUIREMENTS  FOR  CERTAIN  ORGANISMS 

Apparatus.     Sixteen  tubes  of  dextrose  broth. 

Cultures.  Sacch.  cerevisice;  B.  subtilis;  Oospora  lactis; 
Bad.  aerogenes. 

Method.  1.  Inoculate  four  tubes  of  dextrose  broth  with 
each  organism. 

2.  Place  one  culture  of  each  organism  at  each  of  the 
following  temperatures:  5°,  25°,  37°,  and  45°. 

3.  Note  the  growth  as  to  vigor  after  twenty-four,  forty- 
eight,  seventy-two  hours  and  seven  days.    Tabulate  the  data. 

4.  What  is  the  natural  habitat  of  each  organism?     Does 
this  explain  your  results  in  any  way? 

What  is  the  biological  significance  of  the  cardinal  points 
of  temperature?  In  what  industries  making  use  of  micro- 
organisms is  the  regulation  of  temperature  especially 
important? 


EFFECT  OF  FREEZING  ON  BACTERIA      199 

What  inter-relations  have  the  optimum,  minimum  and 
maximum  temperature  requirements  of  one  species  of 
microorganism? 

What  influence  will  the  reaction  of  the  medium  have 
upon  the  extremes  of  temperature  at  which  microorganisms 
will  grow? 

5.  Discuss  your  results  in  detail  and  draw  any  conclu- 
sions permitted.  Point  out  any  practical  applications. 

REFERENCES 

MARSHALL:  Microbiology,  pp.  153-157. 
LAFAR:  Technical  Mycology,  Vol.  I,  pp.  58-60. 
JORDAN:  General  Bacteriology,  4th  Ed.,  pp.  70-72. 

EXERCISE  22.  THE  EFFECT  OF  FREEZING  UPON 
SPORE  -  FORMING  AND  NON  -  SPORE  -  FORMING 
BACTERIA 

Apparatus.  Small  ice  dish;  thermometer;  three  tubes 
each  of  sterile  cider,  sterile  milk,  sterile  broth  and  sterile 
wort;  coarse  salt;  ice. 

Cultures.  Sacch.  cerevisice;  Bad.  lactis  atidi;  B. 
megaterium;  Aspergillus  niger. 

Method.  1.  Heavily  inoculate  the  cider  tubes  with  the 
yeast,  the  milk  tubes  with  Bad.  ladis  acidi,  the  broth  tubes 
with  B.  megaterium  and  the  wort  tubes  with  Aspergillu  niger. 

2.  Incubate  one  set  of  cultures  at  25°  C. 

3.  Make    sufficient    freezing   mixture    of    ice    and    salt 
to  nearly  fill  the  ice  dish. 

4.  Carefully  insert  the  two  remaining  sets  of  cultures 
in  the  freezing  mixture  and  keep  the  freezing  mixture  at  or 
below  0°  C.  for  two  hours. 

5.  Remove  one  set  of  tubes  and  incubate  at  25°  C. 

6.  Then  place  the  ice  dish  containing  the  third  set  of 
cultures  in  the  refrigerator  (note  the  temperature) . 

7.  Examine  both  sets  of  cultures  at  the  end  of  twenty- 
four  hours  and  forty-eight  hours  for  growth. 


200  GENEKAL  MICROBIOLOGY 

8.  Compare   the   three   sets  of  cultures   and  note  the 
variations  from  the  normal  type  of  growth.     Tabulate  your 
data. 

9.  Are   all   of  these   organisms    pecilothermic?      What 
are  termed  the  cardinal  points  of  temperature  for  micro- 
organisms?    What    is    the    lowest    temperature    at    which 
growth,  even  of  the  feeblest  kind,  is  possible?     What  term 
is  applied  to  organisms  which  grow  best  at  low  tempera- 
tures? 

10.  Give  all  results   and  answers  in  full  and  draw  any 
conclusions   permissible.     Point  out  the  practical  applica- 
tions that  may  be  made. 

REFERENCES 

FISCHER:  Structure  and  Functions  of  Bacteria,  pp.  73-75. 
MARSHALL:   Microbiology,  pp.  154-155,  158-159,  199,  318,  395-401. 
LAFAR:  Technical  Mycology,  Vol.  I,  pp.  58-60. 

EXERCISE  23.  THE  DETERMINATION  OF  THE  THER- 
MAL DEATH  POINT  OF  A  SPORE-FORMING  AND  A 
NON-SPORE-FORMING  ORGANISM 

Apparatus.  Water  bath;  test-tube  rack  to  fit  bath; 
ring  tripod;  Bunsen  burner;  thermometer;  thirteen  test 
tubes  of  uniform  diameter  containing  exactly  10  c.c.  of 
broth;  platinum  loop  4  mm.  in  diameter;  dish  containing 
cold  water  (20°  G.  or  below). 

Cultures.  Twenty-four  to  thirty-six  hour  broth  cultures 
of  B.  typhosus  and  B.  mycoides. 

Method.  1.  Examine  the  cultures  for  the  presence  or 
absence  of  spores. 

2.  Set  up  the  water  bath  on  the  ring  tripod,  place  only 
sufficient  water  in  it  to  cover  the  medium  in  the  test  tubes 
and  insert  the  test-tube  rack. 

3.  Insert  the  thermometer  into  one  of  the  test  tubes 
of  broth,  passing  it  through  the  cotton  plug. 

4.  After  flaming  the  plugs,  place  all  the  remaining  tubes 


THERMAL  DEATH  POINT  201 

of  broth  in  the  rack  in  the  water  bath  and  heat  slowly  until 
the  thermometer  in  the  tube  of  broth  registers  45°  C. 

5.  Hold  at  this  temperature  for  fifteen  minutes. 

Note.     Slow   heating  is   necessary  in   order   that   the   respective 
temperatures  may  be  held  for  the  desired  period  of  time. 

6.  Without  removing  the  tubes  from  the  bath,  inoculate 
one  tube  of  broth  witfy  a  loopful  of  the  broth  culture  of 
B.  typhosus,  a  second  with  B.  mycoides.     Carefully  mix  the 
inoculum  with  the  broth  without  removing  the  tubes.     Mark 
each  tube  carefully. 

7.  Allow  these  inoculated  tubes  to  remain  in  the  water 
bath  at  45°  C.  for  ten  minutes. 

8.  Remove  and  place  immediately  in  cold  water. 

9.  Incubate  each  organism  at  its  optimum  temperature 
after  each  trial. 

10.  Next,  raise  the  temperature  of  the  bath  five  degrees, 
i.e.,  to  50°  C.  and  inoculate  the  tubes  as  before  with  B. 
typhosus  and  B.  mycoides. 

11.  Keep  the  tubes  at  50°  C.  for  ten  minutes,  remove 
them  from  the  bath,  cool  and  incubate. 

12.  In  the  same  manner  expose  the  organism  to  the 
following  temperatures,  55°,  60°,  65°  and  70°,  for  a  period 
of  ten  minutes  each. 

13.  In  all  cases  incubate  seven  days  and  record  as  the 
thermal  death  point  (t.  d.  p.)   the  lowest  temperature  at 
which  growth  fails  to  appear. 

14.  What  are  the  standard  methods  for  the  determina- 
tion of  the   t.  d.  p.?      What  are  the  flaws  in  the  above 
method?     What  different  factors  may  influence  the  thermal 
death  point  of  an  organism? 

Do  all  organisms  possess  the  same  t.  d.  p.?     Explain. 

15.  Give  data  and  results  in  full.     Draw  any  conclusions 
that  properly  follow  and  point  out  any  practical  applica- 
tions. 


202  GENERAL  MICROBIOLOGY 

REFERENCES 

ROSENAU:  Preventive  Medicine  and  Hygiene,  pp.  780-781. 
JORDAN:  General  Bacteriology,  4th  Ed.,  pp.  36-37,  72. 
MARSHALL:  Microbiology,  pp.  159-161. 
NOVY:  Laboratory  Work  in  Bacteriology,  pp.  513-518. 

EXERCISE  24.     TO  DETERMINE  THE  RELATIVE  EFFECT 
OF   MOIST  AND   DRY  HEAT   ON   BACTERIA 

Apparatus.  Ten  tubes  of  nutrient  broth  (large  tubes); 
ten  sterile  Esmarch  dishes;  ten  sterile  (flamed)  cover-glasses; 
autoclav;  steam  sterilizer;  hot-air  sterilizer. 

Cultures.  Agar  culture  of  a  spore-forming  organism 
(having  spores  at  the  time). 

Milk  culture  of  slimy  milk  organism,  non-spore-forming. 

Method.  1.  Make  thick  smears  of  each  organism  on  five 
cover-glasses. 

2.  Place  each  cover-glass  of  the  separate  cultures  in  a 
sterile  Esmarch  dish  and  mark. 

3.  Place  two  Esmarch  dishes  of  each  culture  in  the  hot- 
air  sterilizer;   heat  to  120°  C.  and  remove  one  dish  of  each 
culture  after  ten  minutes  at  120°  C.,  the  other  two  after 
thirty  minutes. 

4.  Place  two  smears  of  each   organism  in  the  steam 
sterilizer;,  remove  one  of  each  after  ten  minutes,  the  two 
remaining  after  thirty  minutes. 

6.  Place  the  two  remaining  Esmarch  dishes  in  the  auto- 
clav and  heat  for  ten  minutes  at  120°  C. 

6.  When  cool,  transfer  each  of  the  cover-glasses  to  a 
tube  of  sterile  broth;  mark  carefully. 

7.  Note  in  which  tubes  growth  appears. 

8.  What  is  one  of  the  most  necessary  factors  for  the 
prompt  destruction  of  microorganisms  by  heat?     Why? 

Not  considering  moisture,  what  various  conditions  in- 
fluence the  destruction  of  microorganisms  by  heat?  How 
are  molds  and  yeasts  influenced  by  moist  and  dry  heat? 

To  what  factors  are  the  greater  destructive  powers  of 
the  autoclav  due? 


PASTEURIZATION  203 

9.  Give  all  data  and  results  in  full.     Draw  any  conclu- 
sions possible  and  point  out  any  practical  applications. 

REFERENCES 

HITE,  B.  H.,  GIDDINGS,  N.  J.,  and  WEAKLEY,  CHAS.  E.:  The  effect  of 
pressure  on  certain  microorganisms  encountered  in  the  preservation 
of  fruits  and  vegetables.  Bui.  146,  W.  Va.  Univ.  Agr'l  Expt.  Sta., 
1914. 

MARSHALL:  Microbiology,  pp.  159-161. 

LAFAR:  Technical  Mycology,  Vol.  I,  pp.  79-84. 

FISCHER:  Structure  and  Functions  of  Bacteria,  pp.  75-77. 

EXERCISE  25.  TO  DETERMINE  THE  EFFECT  OF  PAS- 
TEURIZATION UPON  THE  GROWTH  OF  MICRO- 
ORGANISMS 

Apparatus.  300  c.c.  each  of  milk  (not  sterile)  and  of 
some  fermenting  fruit  juice;  water  bath;  two  thermometers; 
four  sterile  200  c.c.  Erlenmeyer  flasks;  twenty-four  tubes 
of  dextrose  agar;  dilution  flasks;  twenty-four  sterile  Petri 
dishes;  sterile  1  c.c.  and  10  c.c.  pipettes. 

Method.  1.  Place  150  c.c.  of  milk  in  each  of  two  200  c.c. 
sterile  Erlenmeyer  flasks;  do  the  same  with  the  fruit  juice. 

2.  Make  three  dilution  plates  each   (1-100,    1-10,000, 
1-1,000,000)  from  the  milk  and  from  the  fruit  juice  in  agar 
and  incubate  (inverted)  at  room  temperature. 

3.  Place  a  flask  of  each  nutrient  liquid  in  the  water  bath 
(cold  water)  and  heat  rapidly  to  75°-80°  (thermometer  in 
each  flask),  shaking  the  flasks  frequently  to  obtain  an  even 
temperature  throughout  their  contents. 

4.  Remove  the  flasks  when  the  temperature  reaches  80° 
C.  and  cool*  them  quickly. 

5.  Make   dilution   plates    (1-10,    1-1,000,    1-10,000)   in 
agar;   mark  each  carefully.     Place  the  flasks  and  plates  at 
room  temperature. 

6.  Place  the  other  two  flasks  in  the  water  bath  (in  cold 
*  It  has  been  found  by  experiment  that  the  quick  cooling  must  take 

place  through  the  temperatures  40°-36°  C.  in  order  to  be  most  efficient 
in  preventing  further  bacterial  growth. 


204  GENERAL  MICROBIOLOGY 

water)  and  heat  slowly  up  to  60°  (thermometer  in  each  flask). 
Keep  at  60°-65°  for  twenty  minutes. 

7.  Remove  the  flasks  from  water  bath  and  cool*  quickly. 

8.  Make   dilution   plates,   using  the  same   dilutions   as 
before,  and  place  the  flask  and  plates  at  room  temperature, 
marking  each  carefully. 

9.  .Watch  daily  for  signs  of  growth  in  each  medium. 

10.  Make  plates  from  each  flask  after  six  days,  deter- 
mining the  range  of  dilutions  by  consulting  your  former 
plates.     Will  the  organisms  have  increased  or  decreased  in 
this  time?     W^hy? 

11.  Compare  the  types  of  organisms  on  the  plates  before 
and  just  after  pasteurizing  and  six  days  after  pasteurizing. 
Examine   each   type   microscopically.     Of   what   does   the 
predominant    flora   of   each    nutrient    fluid    consist    before 
pasteurization?     After  pasteurization? 

Note.     The  fruit  juice  may  be  saved  for  the  experiment  on  meta- 
biosis. 

12.  Count  each  set  of  plates  and  record  the  average 
number  of  microorganisms  per  c.c. 

13.  Plot  the  curve  to  show  the  destruction  of  micro- 
organisms by  pasteurization. 

Compare  the  milk  data  with  milk  data  and  also  the  cider 
with  cider. 

14.  Keep  the  original  flasks  for  one  or  two  weeks.     If 
any  marked  changes  occur,  plate  qualitatively  and  ascer- 
tain the  type  of  organism  causing  the  change. 

15.  How  does  the  physical  nature  of  the  two  nutrient 
substances    influence    their    response    to    pasteurization? 
Give  reasons  for  explanations  offered. 

What  changes  are  brought  about  in  milk  by  pasteuriza- 
tion?    In  cider  or  other  fermenting  fruit  juice? 

16.  Give  all  data  and  results  in  full  and  draw  any  conclu- 
sions.    Point  out  any  practical  applications  that  may  be 

made. 

*  See  note  on  page  203. 


REACTION  OF  THE  NUTRIENT  MEDIUM          205 


REFERENCES 

MARSHALL:  Microbiology,  pp.  319-320,  386-388. 

RUSSELL  and  HASTINGS:  Experimental  Dairy  Bacteriology,  pp.  89-91. 

LAFAR:  Technical  Mycology,  Vol.  II,  Part  I,  pp.  142-144. 


EXERCISE  26.  TO  ILLUSTRATE  THE  EFFECT  OF  THE 
REACTION  OF  THE  NUTRIENT  MEDIUM  UPON 
MICROORGANISMS 

Apparatus.  One  liter  of  ordinary  broth  (should  be 
enough  for  three  students);  normal  NaOH;  normal  acid; 
four  sterile  1  c.c.  pipettes;  10  c.c.  pipettes;  sterile  test 
tubes. 

Cultures.  B.  prodigiosus  (broth  culture);  B.  subtilis 
(broth  culture);  Oospora  lactis  (broth  culture);  Torula 
rosea  (broth  culture). 

Method.  1.  By  adding  normal  acid  or  alkali  produce  in 
100  c.c.  portions  of  ordinary  broth  the  following  reactions: 
-40,  -30,  -20,  -10,  0,  +10,  +20,  +30,  +40,  +50 
degrees  Fuller's  scale,  and  titrate  after  readjusting  the 
reaction,  as  a  check. 

2.  Tube,  using  9.9  c.c.  in  each  tube   (mark  the  tubes 
plainly),  and  sterilize  (refiltration  may  be  necessary  before 
tubing  in  some  cases). 

3.  Using  a  sterile  1  c.c.  pipette,  inoculate  one  set  (ten 
tubes)  with  0.1  c.c.  of  the  broth  culture  of  each  of  the  above 
organisms    (four   sets)    and   incubate   the   tubes   at   room 
temperature. 

4.  Examine  the  tubes  as  often  as  possible  for  the  first 
twenty-four  to  thirty-six  hours,   and  record   the  tube  or 
tubes  in  which  macroscopic  growth  is  first  visible.     What 
do  you  conclude  as  to  the  effect  of  the  reaction  of  the  medium 
in  these  instances? 

5.  Examine  the  tubes  every  day  for  seven  days.     Tabu- 
late  your   observation.      Note    the    range   of   reaction   in 
which  each  organism  is  capable  of  growing.      Does  this 


206  GENERAL  MICROBIOLOGY 

range  differ  with  different  organisms?    Explain  the  action 
occurring. 

6.  In  each  case  inoculate  heavily  the  first  tube  at  either 
or  both  extremes,  in  which  the   organism  fails  to  grow, 
from  the  tube  just  next  it  in  series  which  shows  growth. 
Does  this  freshly  inoculated  tube  show  signs  of  growth  after 
twenty-four  to  forty-eight  hours?     Explain  the  action  which 
occurs. 

7.  Which  organisms  are  acidophiles? 

What  is  the  optimum,  the  minimum  and  the  maximum 
reaction  for  each  organism  according  to  this  experiment? 

What  factors  not  considered  in  this  experiment  might 
influence  results? 

How  would  you  determine  the  exact  optimum  reaction 
of  an  organism? 

8.  Give  all  data  and  results  in  full  and  draw  conclusions. 
Point  out  any  practical  applications. 

REFERENCES 

MARSHALL:  Microbiology,  pp.  173-180,  235-237. 
EYRE:  Bacteriological  Technic,  2d  Ed.,  pp.  305-306. 

EXERCISE  27.     TO   DETERMINE  THE  INFLUENCE   OF 
DIFFUSED   LIGHT   ON   MOLDS 

Apparatus.     Sterile  deep  culture  dish;   tube  of  dextrose 
agar  or  gelatin;  black  paper. 
Culture.     Rhizopus  nigricans. 
Method.     1.  Pour  a  tube  of  agar  into  a  deep  culture  dish. 

2.  When  solid,  inoculate  with  Rhizopus  nigricans. 

3.  Wrap  the  dish  closely  in  black  paper  so  that  no  light 
can  penetrate. 

4.  Cut  a  hole  2  to  3  cm.  in  diameter  in  the  top  edge  of 
the  paper  and  place  the  dish  in  a  north  window  so  that 
only  diffused  light  will  enter  the  aperture. 

5.  Allow  the  dish  to  stand  ten  days,  then  examine  it. 
How  does  diffused  light  influence  this  mold? 


DIFFUSED   LIGHT  ON  MOLDS 


207 


6.  What  is  the  term  applied  to  this  type  of  action? 
How  are  mold  spores  influenced  by  light?     What  influence 
does  diffused  light  have  on  other  microorganisms?     Is  it 
to    be    expected    that    other  common  molds,  Penicillium, 
Oospora,  Aspergillus,  etc.,  would  exhibit  this  same  phenome- 
non? 

7.  Give  all  data  and  observations  in  detail.     Draw  any 


FIG.  44. — Phototropism  Exhibited  by  Rhizopus  nigricans.  The 
mold  was  grown  on  gelatin  with  diffused  light  coming  from  the 
right  side.  (From  Marshall.) 

conclusions  that  follow  and  point  out  any  practical  applica- 
tions. 

REFERENCES 

MARSHALL:  Microbiology,  pp.  162-165. 
JORDAN:  General  Bacteriology,  4th  Ed.,  p.  73. 


208  GENERAL  MICROBIOLOGY 


EXERCISE  28.  TO  SHOW  THE  INFLUENCE  OF  DIRECT 
SUNLIGHT  UPON  THE  GROWTH  OF  MICRO- 
ORGANISMS 

Apparatus.     Two  tubes  of  sterile  agar;   two  sterile  Petri 
dishes;    two  sterile   1   c.c.   pipettes;   two  tubes  of  sterile 


FIG.  45.— Action  of  Direct  Sunlight  on  Bacteria.  These  plates  were 
heavily  inoculated  with  R.  coli  and  B.  prodigiosus,  respectively, 
and  then  were  exposed  bottom  side  up  to  the  direct  rays  of  the 
January  sun  for  four  hours.  At  the  moment  of  exposure  the 
figure  0,  cut  from  black  paper,  was  pasted  to  the  plate,  shading 
the  bacteria  underneath.  After  one,  two  and  three  hours  the 
corresponding  figures  were  pasted  to  the  plates.  The  above 
picture  was  taken  twenty-four  hours  after  exposure,  proving  that 
three  or  four  hours'  exposure  to  direct  sunlight  weakens  and  may 
even  kill  bacteria.  B.  prodigiosus  proved  more  sensitive  than 
B.  coli.  (From  Marshall.) 

distilled  water,  salt  solution  or  broth  for  dilution  purposes; 
black  paper;  glue. 

Cultures.     Ps.  campestris;  B.  typhosus. 

Method.  1.  Inoculate  a  tube  of  sterile  liquid  heavily 
with  Ps.  campestris.  Mix  the  contents  well. 

2.  Place  1  c.c.  of  this  suspension  in  a  sterile  Petri  dish 
and  pour  the  plate. 


SUNLIGHT  UPON  MICROORGANISMS  209 

3.  Duplicate  with  the  B.  typhosus  culture,  placing  the 
pipettes  immediately  after  using  in  1-1,000  HgCb. 

4.  Cut  any  design  out  of  black  paper  and  paste  on  the 
bottom  of  the  Petri  dish. 

5.  Place  the  dish  bottom  side  up  in  direct  sunlight  for 
two  hours. 

6.  Set  the  dish  away  in  the  dark  at  room  tempera- 
ture.    Observe  the  growth  and  explain.     Which  organism 
is  the  more  sensitive  to  sunlight?     Conclusions? 

Note.  Heat  B.  typhosus  plate  1  hour  in  steam  before  cleaning  the 
Petri  dish! 

7.  What  theories  have  been  advanced  as  to  the  mechan- 
ism of  destruction  by  direct  sunlight?     Does  sunlight  have 
any  effect  on  bacterial  spores? 

How  are  other  forms  of  organisms  affected  by  light? 

What  is  phototaxis?  Do  bacteria  ever  exhibit  this 
phenomenon? 

Which  portion  of  the  spectrum  is  most  active? 

What  relation  does  the  wave-length  of  light  rays  bear 
to  the  activity  of  the  rays? 

How  do  diffuse  light,  electric  or  other  forms  of  artificial 
light,  X-rays,  radium  rays,  etc.  compare  with  direct  sun- 
light as  to  their  action  on  bacteria  in  general? 

8.  Give  all  data  and  state  results  in  full.     Draw  any 
conclusions  that  follow  and  point  out  any  practical  applica- 
tions. 

REFERENCES 

MARSHALL,  C.  E.:  Microbiology,  pp.  162-163. 

SMITH,  ERWIN  F. :    Bacteria  in  Relation  to  Plant  Diseases,  Vol.  I, 

p.  71;  Vol.  II,  p.  324. 
LAFAR,  F,;  Technical  Mycology,  Vol.  I,  pp,  60-63, 


210  GENERAL  MICROBIOLOGY 


EXERCISE     29.     DETERMINATION     OF    THE    PHENOL 
COEFFICIENT  OF  SOME  COMMON  DISINFECTANTS 

(Two  students  working  together  are  required  in  this  exercise.) 

Apparatus.  Copper  water  bath;  test-tube  rack  for 
above  bath,  thirty-two  test  tubes  of  uniform  size  containing 
exactly  5  c.c.  of  sterile  nutrient  broth  (use  a  graduated 
burette  or  a  similar  apparatus  for  filling  tubes) ;  eight  clean 
dry  test  tubes  of  uniform  size;  several  (4  or  5)  platinum 
loops,  of  4  mm.  inner  diameter;  sterile  1  c.c.  pipettes  with 
fine  point;  three  clean  5  c.c.  pipettes;  phenol,  5%;  mer- 
curic chloride,  1  :  500;  small  funnel;  filter  paper  to  fit 
funnel;  sterije  test  tube;  watch  with  second  hand. 

Culture.  B.  typhosus,  twenty-four  hour  broth  culture 
grown  at  37°  C. 

Method.  1.  Place  the  filter  paper  in  the  funnel,  wrap  in 
paper  and  sterilize  in  the  hot  air. 

2.  Filter    the    twenty-four-hour    broth    culture    of    B. 
typhosus  into  the  sterile  test  tube.     This  is  for  the  purpose 
of  removing  clumps  of  bacteria  and  any  foreign  matter. 
Funnel  and  filter  paper  are  to  be  treated  immediately  after 
use  with  1  :  1000  HgCl2. 

3.  Regulate  the  water  bath  at  20°  C.  and  keep  at  this 
temperature. 

4.  Mark    the    thirty-two    test    tubes,    each    containing 
exactly  5  c.c.  of  nutrient  broth,  with  the  name  of  the  disin- 
fectant, the  dilution,  and  the  time  exposed,  according  to  the 
following  table.     Then  place  the  tubes,  in  order,  in  the  rack 
in  the  water  bath. 

6.  Mark  each  set  of  clean,  dry  test  tubes  carefully  with 
the  name  of  the  disinfectant  and  the  dilution  to  be  added 
(see  table  on  p.  212),  and  place  in  each,  5  c.c.  of  the  dilu- 
tion of  the  disinfectant  as  indicated  on  the  labels.  Keep  in 
a  test-tube  rack  at  20°  C.  Work  with  one  disinfectant  at  a 
time. 


PHENOL  COEFFICIENT  OF  DISINFECTANTS      211 

N.  B.  Have  the  assistant  carefully  keep  track  of  the  exact 
time  of  all  operations,  to  the  second. 

In  actual  practice  determinations  are  made  oftener  than 
every  five  minutes,  two  and  one-half  minutes  being  the 
standard  interval.  This  requires  the  most  careful  attention 
of  both  operator  and  assistant. 

6.  Using  the  1  c.c.  pipette,  add  0.1  c.c.  of  the  culture  to 
one  tube  of  each  dilution  of  the  disinfectant  and  mix  quickly 
with  a  sharp  rotary  motion  of  the  tube. 

7.  At  the  end  of  one  minute  from  the  time  of  each 
separate  operation,  make  a  loop  transfer  from  the  tube  of 
each  dilution  of  the  disinfectant  inoculated  with  the  cul- 
ture into  the  corresponding  tube  of   broth  in  the  water 
bath. 

Note.  The  assistant  takes  the  tubes  from  the  water  bath  and 
hands  them  to  the  operator,  then,  after  the  operation  of  transferring, 
returns  the  inoculated  broth  tube  to  the  water  bath,  sterilizes  the 
needle  and  places  it  in  the  most  handy  position  for  the  operator.  j 

8.  This  operation  is  then  repeated;   working  as  quickly 
as  possible,  add  0.1    c.c.-  of  the  culture  to  the  remaining 
tubes  of  the  different  dilutions  of  the  disinfectant. 

9.  When,  in  each  case,  the  culture  has  been  exposed  for 
exactly  five  minutes,  ten  minutes  and  fifteen  minutes  respec- 
tively to  the  action  of  the  disinfectant,  a  loop  transfer  is  to 
be  made  to  the  corresponding  tube  of  broth. 

10.  When  all  transfers  are  made,  place  the  broth  cul- 
tures at  37°  C.     Examine  after  forty-eight  hours  for  growth 
and  record  growth  as  +  or  — . 

Note.  The  phenol  coefficient  of  a  disinfectant  is  the  ratio  of  the 
strength  of  the  unknown  disinfectant  which  will  kill  a  filtered  24-  hr. 
broth  culture  of  B.  typhosus  in  a  certain  length  of  time,  to  the  strength  of 
phenol  which  will  accomplish  the  destruction  in  the  same  length  of  time, 
the  dilution  of  phenol  taken  as  1 . 

For  example:  The  dilution  of  an  unknown  disinfectant  required  to 
kill  B.  typhosus  in  7|  minutes  was  1  :  550,  and  the  dilution  of  phenol 
necessary  to  kill  B.  typhosus  in  the  same  time  was  1  :  100.  550-;- 100 
=  5.5,  the  phenol  coefficient  of  the  unknown  disinfectant.  This  means 


212 


GENERAL  MICROBIOLOGY 


that  the  unknown  disinfectant  undiluted  is  5§  times  the  strength  of 
the  undiluted  phenol.* 

11.  Determine    the    approximate    phenol    coefficient    of 
mercuric  chloride  according  to  the  results  of  your  experi- 
ment.    How  does  this  compare  with  results  in  literature? 

12.  What  are  some  of  the  principal  factors  involved  in 
the  examination  of  disinfectants  (pp.  12-20,  Hyg.  Lab.  Bui. 
No.  82).     How  would  each  of  these  come  into  consideration 
in  actual  practice? 

METHOD  OF  MAKING  DILUTIONS  OF  DISINFECTANT  FOR 

TEST 

1  part  of  5%  phenol +1  part  distilled  water  =2.5%  phenol. 
1  part  of  5%  phenol +4  parts  distilled  water  =  1.0%  phenol. 
1  part  of  5%  phenol+9  parts  distilled  water  =  0.5%  phenol. 


1  part  of  1  :  500  HgCl2  +  l  part  distilled  water  =1 
1  part  of  1  :  500  HgCl2+3  parts  distilled  water  =  1 
1  part  of  1  :  500  HgCl2  +9  parts  distilled  water  =  1 


1000  HgCl2. 
2000  HgCl2. 
5000  HgCl2. 


METHOD   OF  RECORDING  RESULTS 


Disinfectant. 


Time  in  minutes  during  which  culture  is  exposed  to 
action  of  disinfectant. 


1  min. 

5  min. 

10  min. 

15  min. 

Phenol  5.0%  
Phenol  2.5%  

Phenol  1.0%. 

Phenol  0.5%  

HgCl2  1     500  
HgCl2  1     1000  

HgCl2  1     2000  

HgCl2  1     5000  

13.  Give  data  and  results  in  full.  Draw  any  conclusions 
that  properly  follow  and  point  out  any  practical  applications. 

*  In  a  large  class  it  would  be  interesting  to  have  determined  the 
phenol  coefficient  of  the  chromic  acid  cleaning  solution  and  of  the  10% 
sodium  hydroxide  used  for  cleaning  glassware,  as  each  is  recommended 
for  immersing  slides,  cover-glasses,  etc.  contaminated  with  bacteria. 


FORMALDEHYDE  UPON  MICROFLORA  OF  MILK    213 


.      REFERENCES 

JOHN  F.  ANDERSON  and  THOMAS  B.  McCLiNTic  :  I.  Method  of  Standard- 

izing Disinfectants  with  and  without  Organic  Matter.     Hygienic 

Laboratory  Bui.  No,  82  (1912),  pp.  1-20,  34,  73. 
S.  RIDEAL  and  E.  K.  RIDEAL:   Some  Remarks  on  the  Rideal-  Walker 

Test  and  the  Rideal-Walker  Method.     Jour,  of  Infectious  Diseases, 

Vol.  X  (1912),  pp.  251-257. 
H.  C.   HAMILTON  and  T.  OHNO:    Standardization  of  Disinfectants. 

Reprint   No.   45    (1913),   from   Research   Laboratory   of   Parke, 

Davis  and  Co.,  pp.  451-458. 
-  The  Bacteriological    Standardization  of    Disinfectants.     Reprint 

No.  65  (1914),  from  Research  Laboratory  of  Parke,  Davis  and  Co. 
M.  L.  HOLM  and  E.  A.  GARDNER:    Formaldehyde  Disinfection  with 

Special   Reference  to   the  Comparative  Value  of  Some  of   the 

Proprietary  Products.     Journal  of  Infectious  Diseases,  Vol.  VII 

(1910),  pp.  642,  643,  650,  658-659. 
MARSHALL:  Microbiology,  pp.  173-180. 

EXERCISE  30.  TO  DETERMINE  THE  ACTION  OF 
FORMALDEHYDE  UPON  THE  MICROFLORA  OF 
MILK 

Apparatus.  Fresh  milk,  skim  or  whole;  1%  solution  of 
formaldehyde;  azolitmin  solution;  twelve  sterile  test  tubes; 
HoS04,  concentrated  commercial. 

Method.  1.  Make  the  following  mixtures  in  sterile 
test  tubes  in  plain  milk,  and  duplicate  in  litmus  milk  (adding 
2%  azolitmin  solution  to  the  milk)  : 

Milk.  Formaldehyde 


9.0  c.c.  +  1  c.c.     ofl%  =0.1% 

9.3  c.c.  +0.7  c.c.  of  1%  =0.07% 

9.7  c.c.  +  0.3  c.c.  of  1%  =  0.03% 

9.0  c.c.  +  1  c.c.    of  0.1%  =  0.01% 

9.0  c.c.  +  1  c.c.     of  0.07%  =  0.007% 

9.0  c.c.  +  1  c.c.    of  0.03%  =  0.003% 

Place  at  room  temperature. 

2.  Record  the  action  in  each  tube,  the  time  required 
for  spoilage  and  the  amount  of  formaldehyde  necessary  to 
preserve  the  milk. 


214  GENERAL  MICROBIOLOGY 

3.  What  is  the  lowest  per  cent  of  formaldehyde  that  has 
inhibitive    action?     That    has    preservative    action?     What 
terms  are    applied    to  these  different  percentages  in  each 
case? 

4.  Make  a  "ring"  test  for    formaldehyde  as    follows: 
Add  several  drops  of  concentrated  commercial  H2SO<i   to 
each  tube  of  plain  milk,  allowing  it  to  run  down  the  side  of 
the  tube  as  in  making  an  ordinary  "  ring  "  test.     A  violet 
coloration  at  the  junction   of  the   H2S04  with  the  milk 
demonstrates  the  presence  of  formaldehyde  in  the  milk. 
The  presence  of  ferric  chloride,  an  impurity  in  commercial 
sulphuric  acid,  is  essential  to  this  test. 

5.  Did  all  percentages  of  formaldehyde  used  give  this 
test?     Did  all  percentages  which  preserved  give  the  test? 

Is  formaldehyde  a  desirable  preservative  for  milk? 
Why?  Are  any  chemicals  more  desirable  for  this  purpose 
than  formaldehyde? 

What  are  the  main  uses  of  formaldehyde?  What  is 
paraformaldehyde?  Its  use? 

6.  State  your  results  in  full  and  draw  any  conclusions 
that  follow.     What  practical  applications  may  be  made? 

REFERENCES 

MARSHALL:  Microbiology,  p.  179. 

SAVAGE:  Milk  and  Public  Health,  pp.  383-391. 

HAWK:  Practical  Physiological  Chemistry,  4th  Ed.,  p.  239. 

EXERCISE  31.     TO  ILLUSTRATE  SYMBIOSIS 

Apparatus.  Sterile  5  c.c.  pipettes;  three  sterile  200  c.c. 
Erlenmeyer  flasks;  450  c.c.  skim  milk;  three  tubes  of  litmus 
milk  (sterile). 

Cultures.     Bad.  lactis  acidi;  Oospora  lactis. 

Method.  1.  Place  150  c.c.  milk  in  each  flask  and  sterilize 
(Tyndall  method). 

2.  Mark  the  flasks,  A,  B  and  C.     Inoculate  flask  A 


MUTUAL  RELATIONSHIP  OF  MICROORGANISMS     215 

with  Bad.  ladis  acidi,  flask  B  with  Bad.  ladis  acidi  and 
Oospora  ladis,  and  flask  C  with  Oospora  ladis  alone. 

3.  Make  ten  titrations,  titrating  every  two  or  three  days 
(not   oftener)    and    record    the   titrations.      Tabulate   the 
data. 

4.  Plot  curves.     How  do  you  explain  the  direction  these 
curves  take? 

5.  At  the  end  of  the  titrations,  make  loop  transfers 
from  each  flask  into  litmus  milk  tubes  and  watch  these 
carefully   in   the   next   twenty-four   to   forty-eight   hours. 
Record  the  results. 

6.  Does  the  action  in  the  flasks  appear  to  be  symbiotic? 
If  so,  how  is  it  shown? 

Is  this  symbiosis  desirable  or  not?     Explain. 
What  other  well-known  examples  of  symbiosis  occur  in 
nature?     Give  a  reason  for  your  statement. 

7.  Give  all  data  and  results  in  full.     Draw  any  conclu- 
sions that  follow  and  point  out  any  practical  operations. 

REFERENCES 

MARSHALL:  Microbiology,  pp.  181-182,  273-282,  323. 

NORTHRUP:    The  Influence  of  Certain  Acid-destroying  Yeasts  upon 

Lactic  Bacteria.     Tech.  Bui.  No.  15,  Mich.  Expt.  Sta.,  pp.  8-16, 

32-34. 

EXERCISE  32.     TO  ILLUSTRATE  ONE  OF  THE  PHASES 
OF  MUTUAL  RELATIONSHIP  OF  MICROORGANISMS 

Apparatus.  Sterile  5  c.c.  pipette;  3  sterile  200  c.c. 
Erlenmeyer  flasks;  450  c.c.  sweet  cider  (that  from  pas- 
teurization experiment  may  be  used). 

Cultures.     Sacch.  ellipsoideus,  Bad.  aceti. 

Method.     1.  Place  150  c.c.  of  sweet  cider  in  each  flask. 

2.  Determine    and    record    the    reaction    of   the    cider, 
then  heat  the  flasks  thirty  minutes  in  the  steam. 

3.  Cool   the  flasks  and  inoculate  flask  A    with  Sacch. 
ellipsoideus. 


216  GENERAL  MICROBIOLOGY 

4.  Determine  the  weight  at  once  and  then  every  day 
until  the  weight  becomes  constant. 

5.  Inoculate  flask  B  with  Bad.  aceti  and  flask  C  with  both 
Bad.  aceti  and  Sacch.  ellipsoideus. 

6.  Titrate  B  and  C  every  two  days.     Titrate  flask  A 
only  at  the  end  of  the  experiment. 

7.  Determine  the  amount  of  alcohol  formed  in  flask  A 


FIG.  46.  Antibiosis.  This  peculiarity  of  growth  is  the  result  of  the 
inhibitive  action  of  metabolic  products  diffused  through  the 
medium.  (Orig.  Northrup.) 

by  distilling  the  contents  of  the  flask  and  determining  the 
specific  gravity  of  the  distillate. 

How  much  CO2  was  given  off?  Calculating  from  this 
amount,  how  many  grams  of  sugar  (CeH^Oe)  were  present 
in  the  flask?  What  percent  sugar  was  this  solution? 

What  was  the  theoretical  amount  of  alcohol  present? 

8.  Plot  curves  showing  the  acid  formation  in  each 
case.  Explain  the  direction  which  these  curves  take. 


EFFECT  OF  THE  METABOLIC  PRODUCTS         217 

9.  Explain  the  mutual  action  and  the  changes  which 
occur. 

10.  What   enzymes   are   responsible   for   each   change? 
Write  out  the  chemical  equations  for  each  change,  giving 
enzyme  concerned  in  each  case. 

Was  the  theoretical  amount  of  alcohol  changed  into 
acetic  acid?  Give  a  reason  for  what  really  does  happen. 

What  phase  of  mutual  relationship  is  illustrated? 

What  is  the  classical  example  of  this  type  of  mutual 
relationship? 

11.  Give  data  and  observations  in  full  and  draw  conclu- 
sions.    Point  out  any  practical  applications. 

REFERENCES 

MARSHALL:   Microbiology,  pp.  182-183,  448-458. 
LAFAR:    Technical  Mycology,  Vol.  I,  pp.  297-307;  Vol.  II,  Part  2, 
pp.  473-481,  511-515. 

EXERCISE  33.  TO  DEMONSTRATE  THE  EFFECT  OF 
THE  METABOLIC  PRODUCTS  OF  BACT.  LACTIS 
ACIDI  ON  ITS  ACTIVITIES 

Apparatus.  Two  sterile  200  c.c.  flasks.  200  c.c.  sweet 
skim  milk;  azolitmin  solution;  apparatus  for  titration; 
sterile  dilution  flasks;  sterile  Petri  dishes;  sterile  1  c.c. 
pipettes;  sterile  5  c.c.  pipettes;  sterile  10  c.c.  pipettes; 
ten  to  fifteen  tubes  of  sterile  litmus  milk. 

Culture.     Bad.  lactis  addi  (twenty-four-hour    culture). 

(At  least  two  weeks  should  be  allowed  for  the  completion  of 
this  experiment.) 

Method.  1.  Place  100  c.c.  skim  milk,  +10°  to  +15° 
(record  acidity  before  adding  azolitmin),  in  each  flask  and 
sterilize  by  the  Tyndall  method. 

2.  Mark  the  flasks  A  and  B. 

3.  Inoculate  each  flask  with  a  loopful  of  a  twenty-four- 
hour  milk  culture  of  Bact.  lactis  acidi.     Mix  well  with  a 
needle  and  plate  dilutions  1-10,  1-100,  1   10,000  from  flask 


218  GENERAL  MICROBIOLOGY 

A  for  obtaining   the   initial   number  of  Bad.  lactis  acidi 
introduced  per  c.c. 

4.  Continue  as  follows: 

Flask  A; 

2d    day,   titrate    and    use    dilutions    1-10,000, 

1-100,000  and  1-1,000,000. 
3d   day,  titrate   and   use   dilutions  1-1,000,000, 

1-10,000,000  and  1-100,000,000. 
6th    day,  titrate    and    use    dilutions    1-100,000, 

1-1,000,000  and  1-10,000,000. 
7th    day,    titrate     and    use    dilutions    1-1,000, 

1-10,000  and  1-100,000. 

N.  B.  Shake  the  flask  of  milk  well  each  time  before  titrat- 
ing and  making  dilutions. 

5.  Titrate  and  plate  every  third  day  thereafter,  until  the 
acidity  remains  constant. 

6.  In  flask  B  from  day  to  day  note  in  millimeters  the 
extent  of  the  re-oxidation  of  the  azolitmin. 

7.  Flask  B.  Without  disturbing  the  milk  any  more  than 
necessary,  make  a  loop  transfer  every  day  or  so  for  10  to 
14  days  from  this  flask  into  a  tube  of  sterile  litmus  milk. 

What  occurs  in  each  case?  In  what  respects  does 
flask  B  check  up  with  flask  A?  Give  explanations  for  sim- 
ilarity or  dissimilarity  of  actions  occurring. 

8.  Milk  contains  on  an  average  about  4.5%  lactose. 
Has   this   sugar   been   fermented   entirely   to   lactic   acid? 
Explain  what  really  occurs. 

9.  What  titre  would  milk  containing  5%  lactose  have 
if  this  sugar  were  entirely  changed  to  lactic  acid? 

Does  any  lactic-acid-producing  organism  approximate 
this  reaction  (in  milk)  at  the  height  of  its  activity? 

10.  Give  reasons  for  what  occurs  in  each  flask.     What 
practical  applications  may  this  experiment  have? 

11.  Tabulate  your  results  and  plot  number  and  acidity 
curves.     Explain  these  curves. 


EFFECT  OF  THE  METABOLIC  PRODUCTS         219 

12.  Draw   any  conclusions  that  follow  from  the  above 
and  point  out  any  practical  applications. 

REFERENCES 

RAHN,  O.:  The  Fermenting  Capacity  of  a  Single  Cell  of  Bad.  lactis 

atidi.     Tech.  Bui.  No.  10,  Mich.  Exp.  Sta.,  p.  25,  et  al 
MARSHALL:  Microbiology,  pp.  157,  308-312. 


PART   III 
APPLIED   MICROBIOLOGY 


AIR  MICROBIOLOGY 

EXERCISE    1.     QUANTITATIVE    BACTERIAL    ANALYSIS 

OF  AIR 

Apparatus.  One  carbon  tube,  dia.  15  mm.;  cork  stopper, 
perforated,  to  fit  carbon  tube;  short  piece  of  glass  tubing 
bent  at  right  angles;  sand  which  has  passed  through  a  150 
mesh  sieve;  8-liter  aspirator  bottle  complete  with  rubber 
stoppers  and  glass  tubing;  sterile  test  tubes  *  containing 
10  c.c.  of  sterile  physiological  salt  solution;  sterile  1  c.c. 
pipettes;  four  sterile  Petri  dishes;  four  tubes  of  sterile 
agar  for  plating;  sterile  agar  slants;  tubes  of  sterile  broth; 
tubes  of  sterile  litmus  milk. 

Method.  1.  Prepare  a  sand  filter  aeroscope  by  placing 
a  layer  of  cotton  in  the  bottom  of  the  carbon  tube. 

2.  Upon  this  place  1  cm.  of  sand  which  has  been  run 
through  a  150  mesh  sieve. 

3.  Insert  a  cork  stopper  through  which  is  passed  a  bent 
glass  tube  plugged  at  the  outer  end  with  cotton. 

4.  Sterilize  the  apparatus  in  hot  air  oven. 

5.  Place  8  liters  of  water  in  the  aspirator  bottle  and 
mark  the  level  of  this  amount  of  liquid. 

6.  Adjust  the  delivery  tube  so  that  it  aspirates  one 
liter  of  air  per  minute. 

*  For  convenience  in  shaking  the  sample,  it  is  recommended  to  use 
test  tubes  with  aluminum  screw  caps,  having  cork  packing. 

220 


QUANTITATIVE  BACTERIAL  ANALYSIS  OF  AIR     221 


7.  Attach  the  aeroscope  (lower  end  of  carbon  tube),  to 
the  aspirator  so  that  the  aspirated  air  will  be  filtered  through 
the  sand. 

8.  Remove  the  cotton  plug  from  the  upper  end  of  the 
aeroscope    and    filter    8  liters    of    air    in 
approximately  eight  minutes. 

9.  Using  "  aseptic  "  precautions,  trans- 
fer as  much  sand  as  possible  to  one  of  the 
tubes  of  sterile  salt  solution. 

10.  Mix   well   by   bumping    the   tube 
against  the  hand  at  least  fifty  times  (do 
not  wet  the  cotton  plug). 

11.  Then,  with  a  sterile  1  c.c.  pipette, 
transfer  1  c.c.  of  the  suspension  to  each 
of  four  Petri  dishes  and  pour  plates. 

12.  Incubate  two  plates  at  37°  C.  for 
two  days,  and  the   remaining   two    plates 
at  room  temperature  for  five  days. 

13.  Count  at  the  end  of  these  respec- 
tive periods  and  determine   the   number 
of  bacteria  per  liter.     How  do  your  counts 
compare    with    air    counts    obtained    by 
other  students?    from  other  data?     (See 
Marshall's  Microbiology,  p.  789.) 

Make  separate   counts  of    molds  and 
identify  them  as  far  as  possible. 

14.  Make    sub-cultures     of    different 
types  on  agar  and   study  their   cultural 
characteristics  on  this  medium. 

15.  Transfer  these  cultures  to  tubes  of 
broth   and   litmus   milk    and    note    their 

action   on    these    media.      Draw    conclusions    from    these 
results. 

16.  What  morphological  types  are  found?     Are  any  of 
the  types  of  bacteria  present  constantly  found  in  air?    What 
are  the  sources  of  microorganisms  in  the  air? 


FIG.  47. — Modified 
Standard  Aero- 
scope. (Ruehle 
and  Kulp.) 


222  GENERAL  MICROBIOLOGY 

Are  any  of  the  types  isolated  related  to  pathogenic  forms? 
May  pathogenic  bacteria  be  isolated  from  air?  If  so, 
under  what  circumstances? 

How  do  microorganisms  enter  the  air?  What  types  of 
microorganisms  are  most  apt  to  be  present  in  air?  What 
is  the  explanation  for  this? 

What  other  methods  may  be  employed  for  obtaining 
quantitatively  the  bacteria  in  the  air? 

Of  what  importance  is  the  quantitative  or  qualitative 
determination  of  microorganisms  in  air? 

17.  Give  data  and  results  in  full  and  draw  any  conclu- 
sions permitted.  Point  out  any  practical  applications  of  the 
above. 

REFERENCES 

RUEHLE,  G.  L.  A.  and  KULP,  W.  L.:  Germ  content  of  stable  air  and 

its  effect  upon  the  germ  content  of  milk,  Bui.  409,  N.  Y.  Expt. 

Sta.,  1915. 

MARSHALL:     Microbiology  (1911),  pp.  185-191. 
EYRE:    Bacteriological  Technic:   2d  Ed.  (1913),  pp.  468-470. 
BESSON:     Practical  Bacteriplogy,  Microbiology  and  Serum  Therapy, 

transl.  by  Hutchens  (1913),  pp.  862-867. 
CHAPIN,  C.  V.:    The  air  as  a  vehicle  of  infection.     Jour.  Amer.  Med. 

Ass'n.,  Vol.  LXII,  pp.  423-430  (1914). 
WINSLOW,-  C.  E.  A.:     Bacteriology  of  air  and  its  sanitary  significance. 

Cent.  f.  Bakt.  Abt.  II.  Bd.,  42,  p.  71  (1914). 
WINSLOW,  C.  E.  A.  and  BROWN,  W.  W.:     The  microbic  content  of 

indoor  and  outdoor  air.     Mo.  Weather  Rev.,  Vol.  XLII  (1914), 

pp.  452-453.     Abst.  in  Exp't.  Sta.  Record,  Vol.  XXXII,  No.  3 

(1915),  p.  211. 


BACTERIOLOGICAL  ANALYSIS   OF  WATER        223 


WATER  AND  SEWAGE  MICROBIOLOGY 

EXERCISE  1.  BACTERIOLOGICAL  ANALYSIS  OF  WATER 
FROM  A  SOURCE  NOT  SUSPECTED  OF  SEWAGE 
CONTAMINATION 

Apparatus.  Sterile  500  c.c.  flask  for  collecting  water 
sample;  litmus  lactose  agar  shake;  twelve  tubes  of  litmus 
lactose  agar;  twelve  salt-free  gelatin  tubes;  two  litmus  lac- 
tose bile  fermentation  tubes;  six  agar  slants;  six  tubes  of 
sterile  broth;  six  tubes  of  Dunham's  solution;  six  tubes  of 
nitrate  peptone  solution;  six  dextrose  fermentation  tubes; 
six  tubes  of  litmus  milk;  twelve  sterile  Petri  dishes;  sterile 
100  c.c.  volumetric  pipette;  sterile  1  c.c.  and  5  c.c.  pipettes; 
record  sheet  for  recording  data  obtained;  record  sheet  for 
recording  pure  cultures  isolated;  water  sample. 

Cultures.     B.  coli. 

Water  from  the  local  water  system  should  be  used  for 
the  experiment.  This  method  can  be  used  also  for  water 
from  deep  wells,  springs,  etc. 

Method.  1.  Flush  the  water  pipes  thoroughly  by 
allowing  the  water  to  run,  or  by  pumping,  at  least  thirty 
minutes. 

2.  Hold  the  collection  flask,  mouth  downwards,  remove 
the  plug  and  still  holding  in  this  inverted  position,  wash 
the  mouth  off  with  the  running  water,  then  fill  quickly 
and  replace  the   plug.     The  plug  must  not  be  laid  down 
during  this  process. 

3.  The  sample  must  be  analyzed  at  once.     In  routine 
work,  if  this  is  not  practicable,  place  the  sample  on  ice 
and  analyze  as  soon  as  possible.     Samples  kept  at  10°  C. 
or  less  should  never  be  left  over  a  maximum  of  six  hours 
before  analysis. 

4.  Plate  immediately  in  duplicate,  1  c.c.,  0.5  c.c.  and 
0.1  c.c.  of  the  sample  direct  in  litmus  lactose  agar  and  in 
gelatin  (6  plates  each).     (If  sewage  contamination  is  sus- 


224 


GENERAL  MICROBIOLOGY 


pec  ted  the  sample  must  be  diluted.     Use  distilled  water 
for  dilution  flasks.) 


FIG.  48. 


FIG.  49. 


FIG.  48.^-A  Model  Dug  Well  Constructed  to  Avoid  Microbial  Con- 
tamination of  Water.  (From  Gerhard's  Sanitation,  Water 
Supply  and  Sewage  Disposal  of  Country  Houses.) 

FIG.  49. — A  Shallow  Driven  or  Tube  Well.     (From  Gerhard.) 


5.  To  the  melted  agar  shake  (at  45°  C.),  add  100  c.c. 
of  the  sample,  using  the  volumetric  pipette,  and  shake  well 


BACTERIOLOGICAL  ANALYSIS  OF  WATER        225 

to  mix  the  sample  thoroughly  with  the  medium.     Avoid 
shaking  so  violently  as  to  produce  gas  bubbles. 

6.  Using  a  sterile  5  c.c.  pipette,  add  5  c.c.  of  the  water 
sample  to  each  litmus  lactose  bile  fermentation  tube. 

7.  Incubate  the  gelatin  plates  (cover-side  up)  in  a  cool 
place. 

8.  Incubate  the  agar  plates  (cover-side  down),  the  agar 
shake  and  the  fermentation  tubes  at  37°  C. 

9.  Make  note  of  the  time  of  day  these  are  placed  in  the 
incubator.     All  cultures  placed  at  37°  C.  must  be  examined 
within  twenty-four  hours.     Types  of  colon-like  organisms  if 
present,  may  be    quite    easily  recognized    within    twenty- 
four  hours  by  the  type  and  reaction  of  the  colony  on  the 
agar  plate,  by  the  fermentation  test,  and  by  acid  and  gas 
formation  in  the  agar  shake. 

10.  Examine  the  agar  plates  after  eighteen  to  twenty- 
four  hours  incubation. 

11.  If  acid   colonies   are   present,   make  morphological 
determinations   (hanging  drop)    for  their  similarity  to  B. 
coli. 

12.  If  these   characteristics   are   positive,   transfer  five 
different  colonies  to  agar  slants. 

13.  Agar  shake  and  fermentation  tubes  must  be  examined 
in  eighteen  to  twenty-four  hours  also  for  gas  and  acid  produc- 
tion. 

14.  If  gas  is  present  in  either,  and  no  acid  colonies  have 
appeared  on  the  original  plates,  make  dilution  plates  in  litmus 
lactose  agar  in  order  to  isolate  the  acid  and  gas  producing 
organisms. 

15.  Transfer  five  different  acid  colonies  which  show  a 
morphology  similar  to  that  of  B.  coli  to  agar  slants. 

16.  At  the  end  of  forty-eight  hours,  remove  the  agar 
plates  from  the  incubator,  make  counts,  record  the  types 
of  colonies  present  and  the  number  of  each  type. 

17.  Transfer  each  type  of  colony  not  previously  isolated, 
to  an  agar  slant. 


226  GENERAL  MICROBIOLOGY 

18.  After  all  agar  slant  cultures  have  grown  sufficiently 
(twenty-four  hours  at  least),  make  sub-cultures  from  each 
pure  culture  into  litmus  milk,  gelatin  stab,  nutrient  broth, 
dextrose   fermentation   tube,    Dunham's   peptone   solution 
and  nitrate  peptone  solution  for  corroborative  tests,  and 
record  the  characteristics  of  growth  according  to  the  descrip- 
tive chart  of  the  Society  of  American  Bacteriologists,   on 
the  form  on  p.  235. 

19.  Determine  to  which  group  of  water  organisms  each 
pure  culture  belongs.     (Consult  the  table  in  Savage's  Bac- 
teriological Examination  of  Water  Supplies,  pp.  192-1S3.) 

20.  Compare  pure  cultures  isolated  from  acid  colonies 
with  a  pure  culture  of  B.  coli  in  each  case. 

21.  Keep  the  plates  at  room  temperature  after  forty- 
eight  hours  of  incubation  and  count  at  the  end  of  seven 
days.     Record  the  counts  as  above. 

22.  Keep  the  agar  shake  and  fermentation  tubes  for 
seven  days  and  record  any  changes  that  take  place. 

23.  Examine  gelatin  plates  after  forty-eight  hours  and 
then  every  day  or  so  for  seven  days. 

24.  Count  before  the  liquefying  colonies  get  so  numerous 
or  large  as  to  render  counting  difficult. 

25.  Record  the  total  number  of  organisms  per  cubic 
centimeter;    also  the  proportion  of  the  liquefying  to  the 
non-liquefying  organisms.     Deep-well  water  should  contain 
none  or  but  very  few  liquefying  organisms.     Why? 

26.  If  liquefying  organisms  are  present  in  large  propor- 
tion or   in  great  numbers   on   the   plates   from   deep-well 
water,  re-plate  from  the  same  source  (not  from  the  original 
sample)    to    determine    whether    the    liquefying    colonies 
came    from  the   original  sample    or   from   some   fault    of 
technic. 

27.  Fish  each  type  of  colony  and  determine  to  which 
group  of  water  organisms  it  belongs.     Are  the  same  organ- 
isms found  on  gelatin  as  on  agar  plates? 

28.  Record  and  compare  the  number  and  types  of  organ- 


BACTERIOLOGICAL  ANALYSIS  OF  WATER        227 

isms  developing  on  the  agar  and  gelatin  plates.     Explain 
why  your  results  vary  on  different  media. 

29.  Does    one    medium    seem   more    favorable    to    the 
development   of   a   larger   number   of   organisms?     If   so, 
which?     Give  reasons  for  answers. 

30.  If  the  gelatin  count  is  less  than  100  organisms  per 
cubic  centimeter  the  water  is  good,  200  per  cubic  centimeter 
will  pass  but  if  many  more,  i.e.,  500,  1000  or  over,  the  water 
is  suspicious  and  effort  should  be  made  to  determine  the 
presence  of  B.  coli. 

31.  Read  the  statements  on  pp.  41-43  and  62-64,  et  al, 
in  Prescott  and  Winslow's  Elements  of  Water  Bacteriol- 
ogy and  compare  with  those  on  p.  77,  Standard  Methods 
of  Water  Analysis.     After  experiments  1  and  2  are  com- 
pleted, draw  your  own  conclusions  from  the  above  and  give 
reasons  for  statements  you  make. 

32.  Each  student  must  know  the  morphological  and  cul- 
tural characteristics  of  B.  coli,  Bad.  aerogenes  and  B.  typho- 
sus. 

Read  about  and  comment  on  the  methods  used  in  other 
countries,  giving  references  to  the  literature  read. 

33.  What   other  methods   are  employed   to   determine 
the  potability  of  water?     Discuss  these. 

REFERENCES 

Standard  Methods  of  Water  Analysis,  American  Public  Health  Asso- 
ciation (1913),  pp.  77-80,  88-96,  el  al. 

PRESCOTT  and  WINSLOW:  Elements  of  Water  Bacteriology,  3d  Ed., 
pp.  1-51,  215-228. 

MARSHALL:     Microbiology,  pp.  192-204. 

SAVAGE:  Bacteriological  Examination  of  Water  Supplies  (1906), 
pp.  192-193,  194-264. 

THRESH:  Examination  of  Waters  and  Water  Supplies,  2d  Ed.  (1913), 
pp.  184-271,  488-524. 

DON  and  CHISHOLM:  Modern  Methods  of  Water  Purification  (1911), 
pp.  260-271, 


228  GENERAL  MICROBIOLOGY 


EXERCISE  2.     BACTERIOLOGICAL  ANALYSIS  OF  WATER 
SUSPECTED  OF  SEWAGE  OR  OTHER  POLLUTION 

Apparatus.  Sterile  500  c.c.  flask  for  collecting  sample; 
litmus  lactose  agar  shake;  six  litmus  lactose  agar  tubes  for 
making  plates;  ten  litmus  lactose  (or  ordinary)  agar  slants 
for  pure  cultures  isolated;  six  salt-free  gelatin  tubes  for 
plates;  ten  tubes  of  gelatin  for  pure  cultures  isolated;  two 
litmus  lactose  bile  fermentation  tubes  (p.  122,  Prescott  and 
Winslow);  ten  tubes  of  litmus  milk;  ten  tubes  of  Dunham's 
peptone  solution;  ten  tubes  sterile  esculin  bile  for  B.  coli 
test  (p.  129,  P.  and  W.);  ten  tubes  of  nitrate  peptone 
solution;  ten  each  fermentation  tubes  of  dextrose,  lactose 
and  saccharose  broth;  99  c.c.  dilution  flasks;  twelve  sterile 
Petri  dishes;  sterile  100  c.c.  volumetric  pipette;  sterile 
1  c.c.  and  5  c.c.  pipettes;  sample  of  water  or  sewage  from 
source  indicated  by  instructor;  record  sheet  for  recording 
data;  record  sheet  for  pure  cultures  isolated. 

Cultures.     B.  coli,  Bad.  aerogenes,  B.  typhosus. 

Method.  Water  for  this  experiment  may  be  obtained 
from  a  lake,  river,  etc.,  just  below  a  sewer  outlet,  or 
from  a  surface  well,  etc.,  the  source  will  be  designated  by 
instructor. 

1.  Collect  the  sample  in  the  sterile  500  c.c.  flask,  using 
all  precautions  as  with  an  unpolluted  water  sample.     (See 
Exercise  1.) 

2.  This  sample  must  be  analyzed  at  once.  . 

3.  Using  a  wide  range  of  dilutions,  plate  immediately 
in  litmus  lactose  agar  and  in  salt-free  gelatin,  making  six 
dilution  plates  in  each  medium. 

4.  If  the  water  is  not  suspected  of  great  pollution,  0.1 
c.c.  of  the  sample  may  be  plated  directly,  using  low  dilu- 
tions for  the  remaining  five  plates. 

5.  If  pure  sewage  is  to  be  plated,  use  dilutions  1  :  100, 
1  :  1000,  1  :  10,000,   1  :  100,000,  1  :  1,000,000  and  1  :  10,- 
000,000. 


BACTERIOLOGICAL  ANALYSIS  OF  SEWAGE      229 


6.  Make  an  agar  shake  as  in  Exercise  1  and  inoculate 
lactose  bile  fermentation  tubes  with  0.1  c.c.  direct  and  1 
c.c.  of  the  1  :  100  dilution  of  the  sample  respectively  (or 

greater  quantities  if  only 
a  small  amount  of  pollu- 
tion is  suspected). 

7.  Incubate  the  agar 
shake,      fermentation 
tubes  and  agar  plates  at 
37°    C.      Place    gelatin 
plates  at  15°  to  20°  C. 

8.  Make  note  of  the 
hour   at  which   the  cul- 
tures are  placed  at  37°  C. 
Examine     these     within 
twenty-four  hours  for  in- 
dications of  the  presence 
of     the     colon-type     of 
organisms. 

9.  Examine  and  count 


FIG.  50. — General  Ground  Plan  of  Individual  House  Sewer  System 
Showing  Exit  from  House,  Anaerobic  Tank,  (A)  Switch,  and 
(B)  Subsurface  Irrigation  Tile. 

the  agar  plates  after  eighteen  to  twenty-four  hours  incuba- 
tion at  37°  C.  (Keep  at  room  temperature  after  forty- 
eight  hours.) 


230 


GENERAL  MICROBIOLOGY 


10.  Fish  acid  colonies,  placing  them  on  agar  slants,  and 
incubate  at  37°  C.  for  twenty-four  hours. 


11.  Examine  each  colony  microscopically  in  a  hanging 
drop.  If  characteristic  of  B.  coli,  make  sub-cultures  in 
litmus  milk,  gelatin  (stab),  Dunham's  solution,  nitrate 


BACTERIOLOGICAL  ANALYSIS  OF  SEWAGE       231 

peptone  solution,  dextrose,  lactose  and  saccharose  fermenta- 
tion tubes,  and  esculin  bile,  for  identification. 

12.  Test  the  gas  for  H  and  CO2  and  record  the  ratio. 


O«=: 

<N 

5 

1 

1 

-,  ,,8,8  

1 

1 

3          Kf)  K^" 

O      rj 

*!•§ 


^       I 
O       I. 


13.  Transfer  B.  coli,   Bad.  aerogenes  and    B.  typhosus 
to  the  same  media  and  compare  their  cultural  character- 
istics with  those  of  organisms  isolated. 

14.  Determine  whether  the  organisms  are  Gram-posi- 
tive or  -negative. 


232 


GENERAL  MICROBIOLOGY 


15.  Examine  gelatin  plates  after  two   days  and  then 
every  few  days  for  seven  days. 


([    i|     r\ 


y 

J9M9S  f 


16.  Count  before  the  liquefying  organisms  get  so  numer- 
ous or  large  as  to  render  counting  difficult. 

17.  Estimate  the  ratio  of  liquefying  to  non-liquefying 


BACTERIOLOGICAL  ANALYSIS  OF  SEWAGE       233 

organisms.     What  is  the  significance  in  sanitary  water  anal- 
ysis of  a  large  number  of  liquefying  organisms? 

18.  If  acid  colonies  are  not  present  in  twenty-four  to 
forty-eight  hours  on  litmus  lactose  agar  plates,  and  acid 
and  gas  are  evident  within  this  time  either  in  the  agar 
shake   or    lactose   bile    fermentation   tubes,  make  dilution 
plates  from  either  of  these  in  litmus  lactose  agar,  incubate 
at  37°  C.  for    twenty-four  hours  and  fish  acid  colonies, 
placing   them   on   the   different   media   for   differentiating 
B.  coli. 

19.  Compare  the  cultures  isolated  with  the  pure  culture 
of  B.   coli  in   every   case.     Also   make   comparisons   with 
Bad.  aerogenes  and  B.  typhosus. 

20.  Isolate  several  different  colonies  and  record  the  cultural, 
etc.,  characteristics  of  each  organism  on  the  record  sheet 
furnished.     Were  any  of  the  types  of  organisms  in  this 
sample  present  in  the  first  sample?     Would  you  expect  to 
find  this  the  case?     Give  reason. 

21.  The  data   and   conclusions  in  the  above  should  be 
given  in  detail.     Point  out  also  any  practical  applications. 

REFERENCES 

Standard  Methods  of  Water  Analysis,  1913  edition,  pp.  79-82,  87-88, 

92,  95-102. 
PRESCOTT  and  WINSLOW:     Water  Bacteriology,  3d.  ed.,  pp.  61-201, 

228-265. 

SAVAGE:     Bacteriological  Examination  of  Water  Supplies,  pp.  27-69. 
MARSHALL:     Microbiology,    pp.   97,    108,    162,    182,    204,    212,    221, 

323-324. 


234 


GENERAL  MICROBIOLOGY 


BACTERIOLOGICAL  WATER  ANALYSIS 

Date .  .          .  .  19 


Sample  No.  . 

Name  of  sender 

Address 

Source  of  water 

Surroundings 

Temperature 

Appearance 

Odor 

Remarks.  . 


Age. 

Agar  Shake. 

Lactose  Bile. 

Per  cent      Per  cent     T>       4  • 
C02.                H2.         Reaction. 

24  hours 

48  hours 

24  hours 

48  hours 

Remarks 

acid 

non- 
acid 

acid 

non- 
acid 

Litmus 
lactose 
agar 
plates 

1.0  c.c. 

0.5  c.c. 

0.1  c.c. 

Average  count 

3  days 

7  days 

liquef. 

non- 
liquef. 

liquef. 

non- 
liquef. 

Gelatin 
plates 

1  c.c. 

0.5  c.c. 

0.1  c.c. 

Average  count 

Plates  made  from  ferm't'n  tube  or  agar  shake .  . 
Organisms  isolated 


] 

Ii 
pSl 

3ACTERIC 

LC 

>GI 

CA 

L 

AS 

L 

ALYS 

1  1 

5lS 

0 

F 

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LTJ 

SB 

235 

s3id  BauuiQ 

80IJM 

Fermentati'on  in 

qiq   ^OBT 

i 

q^oaq  »An 

OSOUS^H 

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uipaj 

^raopy 

__ 

_ 

_ 

— 

^lo^nQ 

asoanqooBg 

1 

aso^OBT  - 

aso^xaa 



— 

— 

i 

ZOO  :  ZH 

1 

eiiq  uiinosa 

•V«ai  ^soaj-aBOA 

_ 

•»S«  -3ua-uoD 

omoj 

— 

3HIUI  -»n 



J13313   '^OBl  •^Ir[ 

q^oaq  paa  ^naN 

JBSB  paa  '^na^ 

u^pa  jo  -bn 

ppuj 

S«nN 

— 

.._ 

— 

_ 

^uauiSijj 

UlB^S  UIBJQ 

_ 

— 

ASopqdao^ 

II 

ii 

|3 

236  GENERAL  MICROBIOLOGY 


EXERCISE  3.  TO  DEMONSTRATE  THE  EFFICIENCY  OF 
CHLORIDE  OF  LIME  AS  AN  AGENT  IN  THE  PURI- 
FICATION OF  DRINKING  WATER 

Apparatus.  Small  can  of  commercial  chloride  of  lime 
(one  can  is  sufficient  for  the  class);  6  liter  precipitating  jar; 
sterile  1.5  liter  flask;  20  sterile  Petri  dishes;  sterile  1  c.c. 
pipettes;  two  sterile  100  c.c.  volumetric  pipettes;  ten  tubes 
of  litmus  lactose  agar;  ten  tubes  gelatin;  two  litmus  lactose 
agar  shakes;  six  dilution  flasks,  four  99  c.c.  and  two  90  c.c, 
(distilled  water) ;  one  liter  of  sewage. 

Method.  1.  Using  the  large  precipitating  jar,  prepare 
a  6%  solution  of  chloride  of  lime  (by  weight),  using  the 
entire  contents  of  a  newly  opened  can.  This  stock  solution 
will  contain  about  2%  available  chlorine  calculated  on  the 
basis  of  35%  available  chlorine  in  the  commercial  chloride 
of  lime. 

2.  Mix  well  and  allow  to  settle  over  night  before  pro- 
ceeding with  the  experiment. 

3.  Obtain  the  sewage  in  the  large  sterile  flask.     Save 
a  small  quantity  of  the  sewage  in  a  sterile  test  tube  for 
microscopic  examination. 

4.  Make  gelatin  and  litmus  lactose  agar  plates  of  the 
fresh  sample,  plating  1  c.c.  and  0.1  c.c.  direct,  and  dilutions, 
1-100,    1-10,000    and    1-1,000,000    (five    plates    in    each 
medium) . 

6.  Add  1  c.c.  of  the  6%  solution  of  chloride  of  lime 
(2%  available  chlorine)  to  exactly  1,000  c.c.  of  the  sewage 
in  the  sterile  flask;  shake  vigorously  for  one  minute  and 
allow  it  to  stand  one  hour.  This  amount  is  over  ten 
times  the  amount  necessary  for  sterilization,  calculating 
on  the  basis  of  16  Ibs.  of  chloride  of  lime  (containing 
35%  available  chlorine)  per  million  gallons  of  water.  (See 
table  on  page  11,  Hooker's  Chloride  of  Lime  in  Sanita- 
tion.) 

6.  Plate  in  gelatin  and  litmus  lactose  agar,  using  1  c.c. 


EFFICIENCY  OF  CHLORIDE  OF  LIME  237 

and  0.1  c.c.  direct,  and  dilutions  of  1-100,  1-10,000  and 
1-1,000,000  (five  plates  for  each  medium). 

7.  Make  duplicate  agar  shakes  also,  with  the  raw  river 
water  and  with  the  same  after  the  chloride  of  lime  has  been 
added. 

8.  Incubate  all  plates  and  shakes  at  37°  C.     Observe 
each  at  the  end  of  twenty-four  and  forty-eight  hours. 

9.  Count  the  agar  plates  at  forty-eight  hours  and  record 
the  results.     Count  the  gelatin  plates  before  the  colonies 
are  obscured  by  liquefiers. 

10.  What   types   of   organisms   have   been   destroyed? 
What  types  remain?     Is  this  according  to  the  results  of 
other  investigators  (see  Hooker,  p.  23).     Would  you  feel 
safe  in  drinking  this  water? 

11.  Examine  the  sewage  in  a  hanging  drop  and  draw 
the  types  of  organisms  present.     Note  which  types  pre- 
dominate, also  note  their  comparative  size  and  motility. 

12.  Make  records  of  these  to  compare  with  the  data  on 
cultural  determinations.     Are  all  of  these  types  found  by 
the  cultural  methods? 

13.  Chloride  of  lime  is  used  for  purifying  drinking  water 
in  the  proportions  of  5  to  25  pounds  per  million  gallons  of 
water.     What  effect  does  chloride  of  lime  have  on  organic 
matter,    discoloration,    turbidity    and    swampy    or    other 
smells  in  raw  water? 

14.  35%    available    chlorine   is   necessary   for   efficient 
sterilization.     The  available  chlorine  is  merely  an  index  of 
the  efficiency  of  the  chloride  of  lime.     Chloride  of  lime  in 
its    industrial    applications    of    bleaching,    deodorizing    or 
disinfecting  does  not  act  by  its  chlorine  but  by  its  oxygen. 
Its    action    is    not    chlorination    but    oxidation.      (Hooker, 
P- 7.) 

15.  What  is  the   maximum  limit   for  the   amount   of 
chloride  of  lime  used  in  dosing  drinking  waters?     What  is 
the  amount  used  for  treating  water  on  shipboard?     Why? 

What  is  the  minimum  length  of  contact  allowed  between 


238  GENERAL  MICROBIOLOGY 

the  stock  solution  of  chloride  of  lime  and  the  water  to  be 
purified?  Why  is  a  minimum  time  limit  set? 

What  other  means  are  used  for  the  chemical  steriliza- 
tion of  water?  Are  these  efficient? 

What  is  the  Hazen  theorem?     Its  explanation? 

What  other  uses  has  chloride  of  lime  in  sanitation? 

16.  Give  all  data  and  observations  in  full.  State  any 
conclusions  to  be  based  on  the  above  and  point  out  any 
practical  applications. 

REFERENCES 

HOOKER:     Chloride  of  Lime  in  Sanitation  (1913),  pp.  7,  12-34,  34-77. 

WESBROOK,  WHITTAKER  and  MOHLER:  The  Resistance  of  Certain 
Bacteria  to  Calcium  Hypochlorite.  Reprint  from  Jour,  of  Amer- 
can  Public  Health  Association,  Vol.  I  (1911). 

WHITTAKER:  Field  Equipment  for  Laboratory  Work  on  Water 
Supplies.  Reprint  from  Journal  of  American  Public  Health  Asso- 
ciation, Vol.  II  (1912),  pp.  948-954. 


EXERCISE  4.  TO  DEMONSTRATE  THE  EFFICIENCY 
OF  THE  BERKEFELD  FILTER  CANDLE  AS  A  MEANS 
OF  WATER  PURIFICATION 

Apparatus.  Berkefeld  filter  candle  complete  with  cylin- 
der; water-power  vacuum  pump;  filter  flask  with  rubber 
stopper  into  which  the  filter  candle  fits;  one  liter  sterile  flask; 
eight  tubes  of  salt-free  gelatin  for  water  analysis;  eight 
Petri  dishes;  sterile  1  c.c.  and  10  c.c.  pipettes;  dilution 
flasks;  distilled  water 

Method.  1.  Set  up  the  filtering  apparatus,  connect 
with  the  vacuum  pump  and  wash  the  filter  by  running 
through  it  500  c.c.  of  boiling,  distilled  water. 

2.  Place  a  cotton  plug  in  the  cylinder  and  in  the  side 
arm  of  the  filter  flask;  sterilize  the  filtering  apparatus  as  set 
up,  in  the  autoclav. 

3.  Collect  a  liter  of  polluted  river  water  from  a  point 
near  the  opening  of  a  sewer. 


TO  TEST  THE  CATALYTIC  POWER  OF  SOIL      239 

4.  Make  dilution  plates  in  gelatin  immediately,  using 
dilutions  1  :  10,  1  :  100,  1  :  10,000  and  1  :  1,000,000. 

6.  Filter  the  remainder  of  the  sample  through  the 
Berkefeld  filter  candle  and  plate  immediately  from  the 
filtrate,  making  gelatin  plates,  using  1  c.c.  and  0.1  c.c. 
direct,  dilutions  of  1  :  100  and  1  :  10,000. 

6.  Incubate  all  plates  at  the  same  temperature.     Ex- 
amine the  plates  daily  and  record  the  counts. 

7.  Was  the  bacterial  count  reduced  by  the  use  of  this 
filter  candle?     What  type  of  organisms  passed  the  filter? 
Is  this  filtered  water  fit  for  drinking  purposes? 

How  does  this  filter  compare  with  other  types  as  to 
its  action?  What  other  types  of  filters  are  used  for  water 
purification?  Sewage  purification?  Upon  what  does  the 
value  of  each  depend? 

8.  Give  all  data  in  full  and  draw  any  conclusions  that 
are  warranted.     Point  out  any  practical  applications. 

REFERENCES 

MARSHALL:     Microbiology,  pp.  205-207. 

DON  and  CHISHOLM:     Modern  Methods  of  Water  Purification  (1911), 
pp.  231-236. 

SOIL  MICROBIOLOGY 

EXERCISE   1.     TO   TEST  THE   CATALYTIC  POWER  OF 

SOIL 

Apparatus.  3%  hydrogen  peroxide;  three  375  c.c. 
Erlenmeyer  flasks;  three  one-hole  stoppers  to  fit  flasks; 
three  pieces  of  bent  glass  tubing;  three  pieces  rubber  tub- 
ing; three  100  c.c.  glass  graduates. 

Culture.     Soil  rich  in  humus. 

Method.  1.  Fit  the  Erlenmeyer  flasks  with  one-hole 
rubber  stoppers  through  which  a  short  piece  of  bent  glass 
tubing  is  inserted.  Fit  short  pieces  of  rubber  tubing 
(about  40  cm.  in  length)  to  the  glass  tubing. 


240 


GENERAL  MICROBIOLOGY 


2.  Place  5  gms.  of  the  soil  in  one  of  the  flasks  and  mix 
it  with  50  c.c.  of  water. 

3.  Arrange  the  100  c.c.  graduate  in  a  water  bath  for 
collecting  gas,  by  filling  with  water  and  inverting  mouth 
down,  under  water.     Clamp  it  in  place. 

4.  Then  add  20  c.c.  of  3%  hydrogen  peroxide  to  the  flask 
containing   the   soil,    stopper   the   flask   and,    keeping   the 
mixture  moving,  collect  the  oxygen  in  the  graduated  tube 
over  water.     Record  the  time  for  maximum  oxygen  libera- 
tion. 

5.  At  the  same  time  determine  the  part  played  by  bac- 
teria and  enzymes,  by  repeating  the  catalase  test  on  soil 
that  has  been  heated  in  the  autoclav  at  15  Ibs.  pressure 
(120°  C.). 

6.  Also  determine  the  part  played  by  humus  by  repeat- 
ing this  test  with  the  same  earth,  having  burned  the  humus 
by  heating  with  the  flame. 

7.  Record  your  results  according  to  the  diagram  follow- 
ing: 


Fresh 
soil. 

Autoclaved 
soil. 

Burned 
soil. 

Cubic  centimeters  of  oxy- 
een  . 

Time  for  maximum  oxy- 
gen liberation  

8.  What   part  does   each    factor  play  in  the   liberation 
of  oxygen?     Is  this  action   in   soil   of  any  value?     If  so, 
what? 

9.  Compare   your   results    with   those   of   the   catalase 
test   in   milk.     By   which   substance  is  the  most    oxygen 
liberated?     Is  there  a  logical  explanation  for  this? 

10.  Give  all  results  in  full,  draw  any  conclusions  that 


TYPES  OF  MICROORGANISMS  IN  SOIL          241 

are  suggested  and  point  out  any  possible  practical  applica- 
tions. 

REFERENCE 

LOHNIS:     Laboratory  Methods  in  Agricultural  Bacteriology,  p.  103. 


EXERCISE  2.  A  COMPARATIVE  STUDY  OF  THE  NUM- 
BER AND  TYPES  OF  MICROORGANISMS  IN  SOIL: 
THE  PLATE  VS.  THE  MICROSCOPIC  METHOD 

A.     THE  PLATE  METHOD 

Apparatus.  Sterile  soil  borer;  sterile  paper  bag;  sterile 
large  spatula;  sterile  large  piece  of  paper;  sterile  paper 
15  cm.  square;  500  c.c.  wide-mouthed  flask  containing  200 


$690,750,000  $1,342,453,982  $2,225,700,000 

Total  Value  of  Total  Value  of  Total  Value  of 

Annual  Timber  Cut.       Mineral  Output,  1911.  Natural  Manure. 

FIG.  54. — Showing  the  Comparatively  Enormous  Value  of  Organic 
Microbial  Food  which  Should  Enter  the  Soil.  (Carver,  Year- 
book of  U,  S.  Dept.  of  Agr.,  1914.) 

c.c.  sterile  water;  90  and  99  c.c.  dilution  flasks  (sterile 
water);  ordinary  agar;  sterile  Petri  dishes;  soil  (different 
types),  manure,  etc. 

Each  student  should  use  one  type  of  soil  and  one  type  of 
manure  for  this  experiment.  Assignments  will  be  made  by 
the  instructor.  The  results  obtained  by  each  student 
are  to  be  compared  with  those  of  others. 


242  GENERAL  MICROBIOLOGY 

Method.  1.  Remove  the  coarser  surface  debris  from 
the  soil.  Sink  the  soil  borer  to  the  depth  of  30  cm.,  remove 
the  borer  and  place  the  soil  in  the  bag. 

2.  Take   six   borings   so   that   your   composite   sample 
will  be  representative  of  the  entire  plot  under  considera- 
tion. 

3.  At  the  laboratory,  carefully  mix  and  pulverize  the 
composite  sample  with  the  spatula  on  the  large  piece  of 
paper. 

4.  Weigh   out  20   gms.   on   sterile   paper  and  transfer 
immediately  to  the  flask  containing  200  c.c.  of  sterile  water. 
A  large  amount  of  soil  is  used  to  reduce  the  error  as  much 
as  possible.     The  manure  should  be  treated  in  a  similar 
manner. 

5.  Shake  thoroughly  for  one  minute,  allow  the  coarser 
particles  to  settle  and  transfer  10  c.c.  (equivalent  to  1  gm. 
of  soil)  of  the  supernatant  liquid  to  90  c.c.  of  .sterile  water. 
Each  cubic  centimeter  of  this  dilution  then  contains  0.01 
gm.  soil. 

6.  Make  and  plate  from  the  following  dilutions:    1-10, 
1-100,  1-1000,  1-10,000,  1-100,000,  1-1,000,000. 

7.  Incubate    at   room    temperature   for    four   to    eight 
days. 

8.  Count  and  record  the  results  as  number  of  bacteria 
per  gram  of  soil  or  manure,  in  tabular  form. 

9.  Record  the  number  of  the  various  types   of   micro- 
organisms.    Note    the    numbers    of    chromogenic    bacteria 
and  the  streptothrix  forms. 

10.  Examine  some  of  the  manure  in  the  hanging  drop. 
What  forms  are  seen?     Make  drawings.     Are  all  of  these 
forms  found  on  the  plates?     Give  reasons  for  what  does 
occur.     Add  sterile  water  to  the  manure  in  a  sterile  deep 
culture  dish  or  flask  and  examine  every  three  or  four  days 
during  the  course  of  the  experiment.     Record  your  obser- 
vations. 

11.  When  the  peat  sample  is  obtained,  at  the  same  time 


TYPES  ^OF  MICROORGANISMS  IN  SOIL  243 

partially  fill  a  small  sterile  flask  with  swamp  or  marsh  water. 
Examine  immediately  in  the  hanging  drop  and  draw  the 
forms  seen. 

12.  Place  this  swamp  water  in  the  sunlight  (more  or 
less  direct)  for  two  or  three  days  and  examine  again  in  the 
hanging  drop  for  any  forms  of  life  present. 

13.  Compare  the  flora  of  these  two  microscopical  prep- 
arations.    Suggest  why  each  type  of  organism  is  present. 

14.  Compare  all  types  of  soil  examined  both  quanti- 
tatively and  qualitatively  as  to  their   microflora.      Which 
soils  are  most  alike  in  their  flora?     Suggest  a  reason  why. 
Why  do  various  soils  vary  in  the  number  of  bacteria  found? 

REFERENCES 

MARSHALL:     Microbiology,  pp.  226-245. 

CONN:     Agricultural  Bacteriology,  pp.  34,  70,  120. 

EYRE:     Bacteriological  Technic.     Second  Ed.,  pp.  470-478. 

B.    MICROSCOPICAL  METHOD 

Apparatus.  Soil  or  manure  of  same  type  as  used  for 
plating;  sterile  water;  sterile  Chinese  ink;  platinum  loop 
of  known  capacity;  sterile  watch  glass;  cover-glasses, 
absolutely  clean]  ocular  micrometer;  stage  (object)  microm- 
eter. 

Method.  1.  To  1  gm.  of  soil  or  excrement  in  a  test 
tube  add  4  c.c.  of  sterile  water  and  shake  vigorously  for 
five  minutes. 

2.  Place  0.5  c.c.  in  a  clean,  sterile  watch  glass.    Add 
0.5  c.c.  of  Chinese  ink. 

3.  Mix  with  a  platinum  loop  of  known  capacity. 

Note.  To  determine  the  capacity  of  the  platinum  loop,  weigh 
two  watch  glasses.  Into  one  put  exactly  1  gm.  (1  c.c.)  of  water. 
Transfer  five  loopfuls  from  the  glass  containing  water  to  the  empty 
watch  glass. 

Weigh  each.  Then  determine  the  weight  and  also  the  volume 
of  one  loopful. 


244  GENERAL  MICROBIOLOGY 

4.  Transfer  one  loopful  of   the  "  ink  manure  "  solution 
to  a  clean,  sterile  cover-glass  and  spread  in  an  even  film 
over  the  entire  surface. 

5.  Let  this  dry  in  air  and  fix  by  passing  three  times 
through  the  flame.     Mount  at  once  in  balsam. 

6.  Measure  the  surface  area  of  the  cover-glass.     Also 
the  diameter  of  one  field  of  the  oil  immersion  lens  (using 
the  stage  micrometer)  and  from  that  the  area  of  the  field. 

7.  Count  fifty  fields  and  determine  the  average. 

8.  From  the  data  which  you  now  have,  determine  the 
number  of  organisms  on  the  cover-glass,  which  is  the  number 
in  one  loopful. 

9.  Then  from  this  calculate  the  number  in  1  gm.  of  soil 
or  excrement. 

10.  Also  calculate  the  weight  of  bacteria  in  1  gm.     (See 
p.  88,  Marshall's  Microbiology.) 

11.  Compare  the  count  thus  obtained  with  the  count 
obtained   by  the  plate  method.     What  is  shown?    How 
do  you  explain  this  result? 

12.  Compare  the  manure  and  soil  counts.     Draw  con- 
clusions and  explain. 

13.  What  other  methods  are  used  for  obtaining  numbers, 
etc.,  of  organisms  in  soil  and  like  materials? 

14.  State  your  data  and  observations   in  full.      Draw 
any    conclusions  warranted  and   point   out  any  practical 
applications. 

REFERENCES 

LOHNIS:     Laboratory    Methods    in    Agricultural     Bacteriology,    pp. 

89-91. 

LIPMAN  and  BROWN:     Laboratory  Guide  in  Soil  Bacteriology,  pp.  7-9. 
MARSHALL:     Microbiology,  pp.  238-244. 


THE  EFFECT  OF  AERATION  OF  SOILS 


245 


EXERCISE  3.  TO  ILLUSTRATE  THE  EFFECT  OF  AERA- 
TION OF  SOILS  ON  THE  ACTIVITIES  OF  THE  MICRO- 
ORGANISMS CONTAINED  THEREIN 

Apparatus.  Coarse,  medium,  and  fine  sand;  magnesium 
oxide;  dilute  HC1;  N/10  NH4OH;  N/10  HC1;  indicator; 
sterile  1  c.c.  pipette;  ten  sterile  250  c.c.  Erlenmeyer  flasks; 
condenser;  sterile  1%  peptone  solution. 

Culture.     B.  mycoides,  broth  culture. 

Method.  1.  Prepare  the  sand  for  use  by  first  heating 
it  with  dilute  HC1;  then  wash  it  several  times,  first  with 
tap  water  then  with  distilled  water  and  dry  at  110°  C. 

2.  Place  50  gms.  of  coarse  sand  in  each  of  three  flasks; 
do  the  same  with  the  medium  and  the  fine  sand. 

3.  Sterilize  the  flasks  in   the  autoclav    at    120°  C.  for 
thirty  minutes. 

4.  Place  50  c.c.  of  the  1%  peptone  solution  in  the  remain- 
ing flask. 

5.  Add  sufficient  peptone  solution  to  the  other  flasks 
to  make  10%,  20%  and  30%  moisture  content  according 
to  the  following  table : 


Soil. 

Moisture. 

After  twenty 
days. 

Coarse  sand  \ 

( 

10% 
20% 
30% 

Medium  sand                                              -\ 

10% 

20% 

30% 

Fine  sand          •! 

10% 

20% 

1%  peptone  solution  

30% 
99% 

6.  Inoculate  each  flask  with  1  c.c.  from  a  broth  culture 
of  B.  mycoides  and  shake  to  distribute  the  organisms  evenly. 


246  GENERAL  MICROBIOLOGY 

7.  Make  ammonia  determinations  after  twenty  days. 

Note.  Ammonia  determinations  from  the  above  are  made  by  the 
following  procedure:  Remove  the  cotton  plugs,  add  200  c.c.  of  water, 
10  gms.  MgO  and  a  small  piece  of  paraffin.  Distill  off  the  ammonia 
present,  collecting  it  in  N/10  HC1  and  titrating  against  N/10  NH4OH, 
us-ing  methyl  orange  as  indicator. 

1      8.  Record  your  results  in  the  form  given  above. 

9.  How   does   aeration   affect   bacterial   activity?     Size 
of  sand  grains? 

What  interrelation  have  grain-size  and  moisture  con- 
tent of  soil? 

How  do  microorganisms  obtain  their  food  in  soil  that 
is  not  wet? 

What  influence  does  humus  have  on  aeration?  On 
bacterial  activity? 

10.  Draw  any  conclusions  warranted    by  your    results 
and  point  out  any  practical  applications. 

REFERENCE 
RAHN,  O.:    Tech.  Bui.  No.  6,  Mich.  Agr'l  Expt.  Sta. 

EXERCISE  4.  TO  DEMONSTRATE  THE  CELLULOSE- 
DECOMPOSING  POWER  OF  AEROBIC  ORGAN- 
ISMS FOUND  IN  THE  SOIL 

Apparatus.  Two  Petri  dishes;  four  pieces  of  round  filter 
paper  to  fit  Petri  dishes;  0.05%  K2HPO4;  MgNH4P04; 
0.05%  NH4NC>3;  1  c.c.  pipette;  soil  rich  in  humus,  or 
well-rotted  manure. 

Method.  1.  Put  a  thin  layer  of  MgNH4P04  between 
two  filter  papers  in  a  Petri  dish. 

2.  Moisten  this  with  the  solution  of  K2HP04  and  in- 
oculate with  a  few  drops  from  a  water  solution  of  the  soil 
or  manure.     Keep  at  25°  to  30°  C. 

3.  In  three  to  six  days  brown  spots  will  occur  and  later 
holes  will  be  formed  by  bacteria.     Thin  places  in  the  filter 


CELLULOSE  DECOMPOSING  POWER  247 

paper  can  be  detected  by  holding  the  Petri  dish  towards  the 
light. 

4.  With  a  sterile  platinum  needle,  test  the  consistency 
of  the  paper  in  the  spots  which  have  been  most  probably 
attacked   and    compare   with   that    of   the   undecomposed 
spots.     Describe  the  results. 

5.  Add  more  of  the  K2HPO4  solution  when  necessary 
to  keep  the  filter  paper  moist. 

6.  Start  a  second  Petri  dish  in  the  same  way  but  keep 
it  moist  with  0.05%  NH4NO3  and  0.05%  K2HPO4.     Here 
we  find  brown  spots  caused  usually  by  fungi. 

7.  Macerate  some  of  the  brown  spots  from  each  Petri 
dish  with  water  and  make  a  Chinese  ink  preparation. 

8.  What  types  of  organisms  are  seen  in  each  prepara- 
tion?    Make  drawings. 

9.  What  organisms  are  especially  active  in  the  anaerobic 
decomposition  of  cellulose?     Which  type,  aerobic  or  anae- 
robic, is  responsible  for   the  greater   amount   of   cellulose 
decomposition  in  nature?    When  may  the  other  types  take 
precedence? 

What  steps  would  you  take  to  isolate  the  organisms 
which  are  growing  on  your  plates? 

Are  the  chemicals  used  above  present  in  the  soil?  In 
what  form?  In  what  forms  does  cellulose  exist  in  culti- 
vated soils? 

Are  cellulose-decomposing  bacteria  ubiquitous?  Are 
they  always  found  where  cellulose  in  some  form  is  depos- 
ited? Are  cellulose  decomposing  bacteria  limited  to  soil? 

10.  Data  and  results  are  to  be  given  in  full,  also  draw  any 
conclusions  warranted  and  point  out  any  possible  practical 
applications. 

REFERENCES 

LOHNIS:     Laboratory    Methods    in    Agricultural    Bacteriology,    pp. 

104-105. 
LIPMAN  and  BROWN:     Laboratory  Guide  in  Soil  Bacteriology,  pp. 

62,  63,  65. 


248  GENERAL  MICROBIOLOGY 

MARSHALL:     Microbiology,  pp.  246-249. 

McBETH  and  SCALES:  The  destruction  of  cellulose  by  bacteria  and 
filamentous  fungi.  Bui.  266,  B.  P.  I.,  U.  S.  Dept.  Agr.  (1913). 

EXERCISE  5.  TO  ILLUSTRATE  THE  ANAEROBIC  DE- 
COMPOSITION OF  CELLULOSE  BY  SOIL  AND 
FECAL  ORGANISMS 

Apparatus.  Six  (large)  tubes  of  Omelianski's  synthetic 
medium  for  anaerobic  cellulose  fermentation;  tall  Novy 
jar;  vacuum  pump. 

Culture.     Fresh  and  decayed  manure. 

Method.  1.  Inoculate  one  tube  with  small  amounts 
of  fresh  horse  or  cow  manure,  a  second  with  partially  de- 
cayed manure. 

2.  Place  some  cotton  in  the  bottom  of  the  Novy  jar, 
insert  the  inoculated  tubes  in  it,  replace   the  stopper  and 
exhaust  the  air  by  means  of  the  vacuum  pump.     (Pyro- 
gallic  acid  and  sodium  hydroxide  may  be  substituted.) 

3.  Incubate  the  tubes  in  the  Novy  jar  at  34°  to  35°  C. 
for  four  to  six  weeks. 

4.  From  time  to  time  note  any  changes  occurring  in  the 
filter  paper. 

5.  After  the  latter  has  been  wholly  or  partially  diges- 
ted, make  transfers  to  new  tubes  of  the  medium  and  incu- 
bate anaerobically  as  before. 

6.  Repeat  this  procedure  from  the  cultures  made  just 
previously.     (The  jar  is  evacuated  each  time  after  obser- 
vations are  made.) 

7.  Examine  the  organisms  causing  the  disintegration  of 
the  filter  paper  both  in  the  hanging  drop  and  with  some 
ordinary  stain  (not  the  ink  preparation).     Make  permanent 
stained  preparations. 

8.  Starch,  cotton,  straw,  etc.,  digestion  may  be  com- 
pared if  these  substances  are  substituted  for  filter  paper 
in  Omelianski's  medium. 

Not  taking  soil  into  consideration,  where  do  anaerobic 


NITRIFICATION  IN   SOLUTION 


249 


cellulose-decomposing    organisms    probably    play    a    most 
important  part?     How  is  this  determined? 

9.  What  types  of  anaerobic  cellulose-decomposing  bac- 
teria are  favored  by  this  synthetic  medium?     These  bac- 
teria have  only  in  exceptional  cases  been  grown  on  solid 
media.     How  can  these  types  be  separated? 

10.  Data   and   observations   should   be   given   in   full. 
Draw  any  conclusions  warranted  and  indicate  any  practical 
applications  that  may  be  made, 

REFERENCES 

LOHNIS:     Laboratory  Methods  in  Agricultural  Bacteriology,  p.  93. 
LIPMAN  and  BROWN:     Laboratory  Guide  in  Soil  Bacteriology,  p.  64. 
MARSHALL:     Microbiology,  pp.  246-248. 
McBETH  and  SCALES:    I.e.,  Exercise  IV. 


EXERCISE  6.     TO  ILLUSTRATE  NITRIFICATION  IN 
SOLUTION 

Apparatus.  Nine  50  c.c.  Erlenmeyer  flasks;  75  c.c. 
each  of  Solutions  I,  II  and  III  for  nitrification  (see  appen- 
dix); Nessler's  solution;  a-naphthylamin;  sulphanilic  acid; 
2%  diphenylamin  in  sulphuric  acid;  aqueous  alcoholic 
stains. 

Cultures.  Rich  soil  from  cultivated  land;  manure 
from  surface  layers  of  manure  heap. 

Method.  1.  Place  25  c.c.  of  each  solution  in  a  50  c.c. 
Erlenmeyer  flask  and  sterilize  in  the  autoclav. 

2.  Inoculate  as  follows: 


Solution  I. 

Solution  II. 

Solution  III. 

0.5  gm.  manure  
0.5  gm.  soil  
Nothing  —  control  

and  keep  at  25°  to  30°  C.,  to  hasten  the  action  of  the  micro- 
organisms. 


250  GENERAL  MICROBIOLOGY 

Owing  to  the  presence  of  some  carbon-monoxide  in  the 
air  of  the  laboratory  from  the  burning  gas,  the  carbon- 
monoxide-assimilating  B.  oligocarbophilus  often  appears  as 
a  dry  white  skin  on  the  surface  of  the  solution  in  these  flasks. 

Note.  Solution  I  is  adapted  for  relatively  increasing  the  nitrite 
bacteria,  Solution  II  the  nitrate  producers  and  Solution  III  the  sim- 
ultaneous growth  of  both  organisms  as  in  nature. 

3.  After  eight  to  fourteen  days,  test  all  solutions  and 
control  flasks  every  second  or  third   day  by  transferring 
0.1  c.c.  with  a  sterile   pipette   to   a  white   glazed   surface 
(e.g.,  plate)  using 

(a)  Nessler's  solution,  for  ammonia; 

(6)  nitrite  test  solutions,  for  nitrites; 

(c)   nitrate  test  solution,  for  nitrates. 

Tabulate  your  results.  Discuss  and  explain  the  decom- 
position which  is  taking  place  in  each  inoculated  flask, 
giving  the  successive  steps  in  the  disintegration  of  the 
crude  nitrogenous  organic  matter. 

4.  Examine  a  loopful  of  each  solution  in  the  hanging 
drop  each  time  chemical  tests  are  made.     Morphologically 
what  types  predominate  in  each  solution?     In  the  sample 
of   soil?     Of   manure?    Are   any   of   these   spore-formers? 
If  so,  which  type? 

5.  Make    permanent    stained    preparations    from   each 
flask. 

6.  Nitrifying   bacteria   do   not   grow   on   the   ordinary 
solid  media.     Why?     Many  different  methods  have  been 
tried  for  the  isolation  of  nitrifying  organisms  but  the  obtain- 
ing of  pure  cultures  is  still  a  most  difficult  bacteriological 
task. 

(See  appendix  for  media  used  for  the  isolation  of  these 
organisms.) 

What  methods  are  employed  for  their  isolation  besides 
the  use  of  solid  synthetic  media?  What  is  the  principle 
of  each  method? 

What  are  the  different  types  of  nitrifying  organisms? 


DENITRIFICATION  IN  SOLUTION  251 

Their  respective  functions?  What  interrelationships  exist 
between  these  organisms? 

Where  are  nitrifying  organisms  found  in  nature?  What 
is  their  significance? 

W^hat  conditions  in  soil  are  necessary  for  their  pro- 
liferation? What  methods  does  the  agriculturist  use  which 
serves  to  conserve  these  organisms? 

REFERENCES 

LOHNIS:     Laboratory    Methods    in    Agricultural    Bacteriology,    pp. 

96,  97,  106,  109,  110. 
LIPMAN  and  BROWN:     Laboratory  Guide  in  Soil  Bacteriology,  pp. 

25-34. 
MARSHALL:     Microbiology,  pp.  259-263. 

EXERCISE    7.     TO    ILLUSTRATE  DENITRIFICATION  IN 
SOLUTION 

Apparatus.  Eight  tubes  of  sterile  Giltay's  solution; 
eight  tubes  of  nitrate  broth;  eight  to  ten  tubes  of  Giltay's 
agar;  eight  to  ten  tubes  of  nitrate  agar;  eight  fermenta- 
tion tubes  of  nitrate  bouillon,  four  with  and  four  .without 
sugar  (use  1%  dextrose);  peptone  solution  in  tubes;  sterile 
1%  solution  of  KNOs;  nitrate  test  solution;  Nessler's 
solution,  test  for  NHs;  a-naphthylamin  and  sulphanilic  acid 
(nitrite  test  solutions);  sterile  1  c.c.  pipettes;  sterile  Petri 
dishes;  sterile  dilution  flasks. 

Cultures.  Soil  (record  type  used);  straw  from  manure 
pile;  horse  manure. 

Method.  1.  Inoculate  test  tubes  of  each  solution  in 
series  as  follows: 

1  and  2— 

3  and  4 — 0.1  gm.  soil. 

5  and  6 — 0.1  gm.  straw; 

7  and  8 — 0.1  gm.  horse  manure. 

2.  Incubate  these  at  37°  C.  for  forty-eight  hours. 

3.  Note    any    changes    occurring.     Determine   whether 


252  GENERAL  MICROBIOLOGY 

any  nitrites  or  ammonia  have  developed.     To  what  are 
the  gas  bubbles  due? 

The  crystals  deposited  in  Giltay's  solution  are  magnesium 
phosphate. 

4.  Using  sterile  pipettes,  test   1   c.c.   portions  of  each 
after  forty-eight  hours,  seven  days,  etc.,  for  nitrates  with 
phenolsulphonic  acid. 

5.  From  two  tubes  showing  abundant  gas  formation, 
make  nitrate  agar  plates,  using  a  wide  range  of  dilutions 
that  one  or  two  plates  may  show  well-isolated  colonies. 
Incubate  at  37°  C. 

6.  From  various  colonies  appearing  on  the  plates,  make 
stab  cultures  in  nitrate  agar.     Incubate  these  at  37°  C. 

Save  one  showing  the  most  abundant  gas  formation  under 
these  conditions,  for  further  study. 

7.  Inoculate  a  nitrate  bouillon  fermentation  tube  with 
the  pure  culture  just  isolated,  also  add  some  of  the  crude 
material  to  a  fermentation  tube. 

8.  Duplicate  with  a  nitrate  bouillon  fermentation  tube 
containing  sugar. 

9.  Determine  the  amount  and  nature  of  the  gas  formed 
in  each  tube  and  compare  results.     (Determine  by  elimi- 
nation;   test  for  CO2  and  H2.) 

What  influence  does  dextrose  have  upon  the  rate  and 
amount  of  gas  formation? 

10.  Inoculate  in  duplicate,   tubes  of  peptone  solution 
with  the  pure  culture.     Record  the  growth  and  gas  forma- 
tion, if  any,  qualitatively. 

11.  To  an  old  culture  (not  necessarily  pure)  in  which  the 
nitrates  have  disappeared,  add  1  c.c.  of  a  sterile  1%  solu- 
tion of  KNOs.     Does  gas  formation  re-occur? 

12.  Continue  to  add  a  small  amount  of  KNOs  as  rapidly 
as  the  culture  ceases  to  give  a  reaction  for  nitrates  (an 
indication  that  the  latter  have  been  used  up.)     Note  how 
much  KNOs  your  culture  can  reduce. 

13.  Theoretically,   what    would    be    the    difference    in 


NON-SYMBIOTIC  FIXATION  OF  NITROGEN        253 

action  of  denitrifying  organisms  in  soil  and  in  solution? 
Why? 

How  do  denitrification  and  nitrate  reduction  differ? 

How  may  colonies  of  nitrate-reducing  bacteria  be 
detected? 

14.  Give  all  results  in  full  and  draw  conclusions.  Sug- 
gest any  possible  practical  applications  of  the  above. 

REFERENCES 

LIPMAN  and  BROWN:     Laboratory  Guide  in  Soil  Bacteriology,  pp. 

35-40. 

MARSHALL:     Microbiology,  pp.  263-267. 
LOHNIS:     Laboratory  Methods  in  Agricultural  Bacteriology,  pp.  98-99. 

EXERCISE  8.  TO  ILLUSTRATE  THE  NON-SYMBIOTIC 
FIXATION  OF  NITROGEN  BY  SOIL  ORGANISMS, 
AND  ISOLATION  OF  AZOTOBACTER  THROUGH 
ITS  MINERAL  FOOD  REQUIREMENTS 

Apparatus:  Mannit  solution  for  nitrogen-fixing  organ- 
isms; mannit  agar;  eight  sterile  100  c.c.  Erlenmeyer 
flasks;  concave  slides;  cover-glasses;  concentrated  H2S04; 
filter  paper. 

Culture.     From  clay  loam,  sandy  loam,  manure. 

Method.  1.  Place  50  c.c.  of  the  mannit  solution  in 
each  flask  and  sterilize  (in  autoclav). 

2.  Inoculate  in  series  as  follows: 
Flasks  1  and  2— 

Flasks  3  and  4 — 0.1  gm.  clay  loam. 
Flasks  5  and  6 — 0.1  gm.  sandy  loam. 
Flasks  7  and  8 — 0.1  gm.  manure. 

3.  Incubate  at  room  temperature  and  note  the  changes 
taking  place.     A  wrkikled  skin,   wnite  at  first,   brownish 
later,  is  gradually  formed.     This  is  composed  of  the  aerobic 
Azotobacter. 

4.  From  time  to  time  examine  the  cultures  in  the  hang- 
ing drop  and  note  the  type  of  organisms  predominating. 
Single,   small,   thin  bacilli  are  visible  between  the  large 


254 


GENERAL  MICROBIOLOGY 


cells  of  Azotobacter.  The  former  are  almost  always  a  type 
resembling  the  nodule  bacteria,  B.  radiobacter,  and  which 
can  also  fix  nitrogen  to  a  slight  extent.  Besides  these, 
especially  when  Azotobacter  is  less  in  evidence,  many  other 
sporing  and  non-sporing  bacteria  participate  in  the  process. 
Azotobacter,  however,  is  the  most  vigorous  free  nitrogen- 
fixing  organism  yet  discovered. 
(See  reference,  E.  B.  Fred, 
Exercise  9,  Soil  Microbiology.) 

5.  What  characteristic  odor 
is  produced  in  these  cultures? 
Add   a   drop   of    concentrated 
H2SO4   to   a   small  portion   of 
the    culture    liquid.     This    in- 
tensifies the  odor. 

6.  When  a  brownish  surface 
film  develops,  make  plates  from 
this    culture,    using    relatively 
high  dilutions. 

7.  After  a  rather  long  period 
of    incubation    (six    to    seven 
days)  examine  the  organisms  in 
the  various  colonies  and  isolate 
Azotobacter  chroococcum  if  possi- 
ble upon  a  mannit  agar  slant. 
On  account  of  the  slimy  prop- 
erty of  its  cell  wall,  its  separa- 
tion from  B.  radiobacter  is  often  very  difficult.     The  quickest 
way  is  to  reinoculate  first  into  the  mannit  solution. 

8.  Save  several  of  the  plates  having  well-isolated  colo- 
nies and  note  any  changes  which  may  occur. 

9.  If  any   brown   colonies   develop,    examine   them   in 
stained  preparations.     Measure  the  bacteria  stained. 

10.  Are  these  pure  cultures?     If  not,  plate  from  several 
such  colonies  in  mannit  agar  to  isolate  the  different  organ- 
isms  present. 


FIG.  55.— Azotobacter.  xlOOO; 
Smear  from  Six-day  Old  Cul- 
ture on  Ashby's  Agar  at 
25°  C.  Showing  organisms 
and  capsules  in  various 
stages  of  development.  (Dan 
H.  Jones.) 


NON-SYMBIOTIC  FIXATION  OF  NITROGEN        255 

11.  Is  B.  radiobacter  present?     What  part  does  it  play 
in  the  fixation  of  nitrogen? 

12.  Make  several  agar  slant  pure  cultures  of  B.  radio- 
bacter. 

13.  Study  the  morphology  and  the  cultural  character- 
istics of  this  organism. 

14.  Inoculate  a  small  flask  of  the  mannit  solution  with 
a  pure  culture  of  the  newly  isolated  organism. 

15.  How  does  this  organism  compare,  morphologically, 
culturally,  etc.,  with  Ps.  radicicolaf     With  the  Azotobacter? 

What  part  in  the  nitrogen  cycle  does  the  Azotobacter  play? 

What  practices  of  the  farmer  favor  the  development  of 
the  Azotobacterf  In  what  way?  What  soil  conditions  are 
favorable  to  the  Azotobacter  species?  Are  these  condi- 
tions favorable  to  other  bacteria?  To  plants? 

Diseased  spots  in  soil  are  said  to  be  caused  by  an 
excessive  nitrogen  fixation  and  nitrification,  e.g.,  the  niter 
spots  in  Colorado  soils. 

16.  State  your  results  in  full  and   draw  conclusions. 
Point  out  the  practical  applications  of  the  above. 

REFERENCES 

MARSHALL:     Microbiology,  pp.  98-99,  230-231,  248,  250,  266,  270-273, 

286,  288,  291. 
LIPMAN  and    BROWN:     Laboratory  Guide  in  Soil  Microbiology,  pp. 

43-45. 
HOFFMAN,  C.,  and  HAMMER,  B.  W.:    Some  Factors  Concerned  in  the 

Fixation  of   Nitrogen  by   Azotobacter.    Research   Bui.   No.    12, 

Univ.  of  Wis.  (1910). 
JONES,   DAN  II.:    A   Morphological  and   Cultural  Study   of  Some 

Azotobacter.     Cent.  f.  Bakt.  II  Abt.,  Bd.  38  (1913),  pp.  13-25, 

5  plates. 
Further  Studies  with  Some  Azotobacter.    Cent,  f .  Bakt.  II  Abt., 

Bd.  42,  pp.  68-69. 
LOHNIS:     Laboratory  Methods  in  Agricultural  Bacteriology,  pp.  40, 

113-114,  127. 
HEADDEN,  W.  P. :    The  Fixation  of  Nitrogen  in  Colorado  Soils.     Buls. 

178,  186,  Colorado  Agr.  Expt.  Sta. 


256  GENERAL  MICROBIOLOGY 

EXERCISE  9.  A  STUDY  OF  THE  SYMBIOTIC  NITROGEN- 
FIXING  ORGANISMS  OF  LEGUMES,  PS.  RADICI- 
COLA 

A.  ISOLATION   OF    PS.   RADICICOLA    FROM 
NODULES  OF  LEGU MINOS  JE 

Apparatus.  Spade  or  trowel;  sterile  Petri  dishes; 
tubes  of  nitrogen-free  ash  agar;  tumbler  for  mercuric 
chloride  solution;  small  piece  of  filter  paper;  small  pair 
of  forceps;  scalpel  or  chisel-edged  platinum  needle;  platinum 
loop;  clean  slides;  mercuric  chloride,  1-500;  alcohol,  95%; 
tubes  of  sterile  water;  aqueous-alcoholic  gentian  violet 
orfuchsin;  eosin;  LugoPs  iodin  solution;  saturated  alcoholic 
solution  of  gentian  violet. 

Culture.     From  nodules  of  roots  of  leguminous  plants. 

Method.  1.  Using  a  spade  or  trowel,  obtain  the  roots 
of  some  legumes  which  show  nodule  formation  in,  abundance. 
If  the  soil  is  firm,  as  with  clay,  do  not  forcibly  pull  up  the 
legume  to  obtain  the  roots  as  this  procedure  strips  off  the 
nodules  which  develope  almost  without  exception  on  the 
young  rootlets. 

Note.  Heavily  inoculated  legumes  may  be  stored  for  winter  use  in  a 
cool,  dry,  dark  place.  Living  organisms  have  been  found  after  more 
than  two  years  in  some  of  the  larger  nodules. 

2.  Thoroughly  wash  the  roots  under  the  tap  and  place 
the  plant  in  clean,  cool  water. 

3.  Keep   parts   of   the   plant   for   identification   if   the 
species  is  unknown.     Not  all  plants  belonging  to  the  family 
Leguminosce    are    attacked     by    symbiotic     nitrogen-fixing 
bacteria,  only  those   belonging   to  the  sub-family,  Papilio- 
nacece. 

4.  Compare  the  size,  numbers,  and  location  of  the  nod- 
ules on  the  roots  of  the  different  legumes. 

5.  Remove  a  good-sized  nodule  from  the  roots,  wash 
in  clean  water  and  immerse  for  three  minutes  in  mercuric 
chloride  solution,  1  :  500. 


NITROGEN-FIXING  ORGANISMS   OF  LEGUMES    257 

6.  Remove  the  nodule  with  flamed  forceps  and  take  up 
the  excess  of  the  solution  between  folds  of  sterile  filter 
paper,   then   dip  into   alcohol,   the  last  traces  of  alcohol 
being  removed  by  passing  the  nodule  quickly  through  the 
flame. 

7.  Place  the  nodule  on  a  flamed  and  cooled  slide. 

8.  Holding  the  nodule  in  flamed  and  cooled  forceps, 
cut  into  it,  and  break  it  open  by  means  of  a  sterile  scalpel 
or  a  chisel-edged  platinum  needle. 

9.  Thrust  a  sterile  platinum  needle  into  the  nodule  in 
the  middle  of  the  newly  exposed  surface  and  gently  rotate  the 
needle  so  that  some  of  the  crushed  tissue  adheres  to  it. 

10.  Touch  the  needle  in  a  drop  of  sterile  water  in  a  sterile 
Petri  dish. 

11.  Transfer  a  loopful  of  this  suspension  to  a  second 
drop  of  sterile  water  in  a  second  Petri  dish  and  a  loopful 
from  this  to  a  third  drop  in  a  third  Petri  dish. 

12.  Pour  the  relates,  using  tubes  of  nitrogen-free  ash 
agar  and  incubate  at  room  temperature  for  two  or  three 
days. 

13.  Make  a  smear  on  a  clean  slide  from  the  freshly  cut 
surface  of  the  nodule,  stain  and  examine  microscopically. 
What  is  the  morphology  of  Ps.  radidcola  found  in   the 
nodule?    What  are  bacteroids? 

14.  Make  a  smear  directly  from  this  same  nodule  on 
a  clean  slide,  fix  and  stain  with  eosin  followed  by  Lugol's 
iodin  solution.     The  iodin  demonstrates  the  starch  which 
is  usually  present  in  nocjules.     Is  starch  present? 

15.  After  a  few  days  of  incubation  the  colonies  of  Ps. 
radidcola  will  be  noted  on  the  plates  as  round,  grayish- 
white,  translucent,  slime-like  drops,  finely  granulated  and 
often  with  compact  white  centers. 

16.  Examine  these  colonies  in  the  hanging  drop.     They 
contain   the   normal,   short,   rod   forms  which   during  the 
first  days  are  very  motile. 

17.  Isolate  several  pure  cultures  of  Ps.  radidcola  on 


258  GENERAL  MICROBIOLOGY 

slanted  nitrogen-free  agar  and  note  characteristic  growth. 
Why  is  nitrogen-free  agar  used  for  the  cultivation  of  Ps. 
radicicolaf 

18.  Make  permanent  stains  of  pure  culture  and  compare 
with  organisms  on  stained  smear  from  nodule  as  to  size, 
shape,  etc.     Are  involution  forms  present  in  either  prepara- 
tion? 

Do  all  species  produce  organisms  of  the  same  general 
morphology  in  the  respective  nodules?  In  pure  culture? 

19.  Make  a  flagella  stain  from  the  pure  culture  as  fol- 
lows: 

a.  Take  a  loopful  of  the  mucilaginous  growth  from  a 
colony  or  an  agar  culture  and  spread  it  on  a  clean  slide, 
lashing  it  out  in  slender  tongues. 

6.  Let  the  film  dry  in  air  without  killing  or  fixing. 

c.  Flood  the  film  a  moment  with  saturated  alcoholic 
solution  of  gentian  violet. 

d.  Wash  under  the  tap,  dry  and  examine  with  the  oil 
immersion  lens. 

20.  The  mucilage  in  which  the  cells  lie  will  be  found 
to  be  deeply  and  evenly  stained  and  the  bacteria  stained 
scarcely  at  all,  so  that  the  preparation  presents  somewhat 
the  appearance  of  a  photographic  negative. 

The  single  polar  flagellum  may  be  demonstrated  by 
this  stain,  since  it,  like  the  protoplasm  of  the  cells,  refuses 
the  stain,  and  so  it  appears  as  a  clear,  uncolored  streak  in 
the  surrounding  deeply  stained  mucilage.  The  flagella 
are  best  seen  at  the  margins  and  in  thin  places,  inasmuch 
as  the  mucilage  in  the  denser  areas  masks  the  slender  fla- 
gella. 

21.  Sometimes  the  roots  of  leguminous   plants  show, 
instead  of  the  normal  nodules,  lesions  of  crown-gall  caused 
by  Bad.  tumefaciens  which  somewhat  resemble  Ps.  radici- 
cola. 

22.  For  a  rapid  diagnosis,  plate  in  the  synthetic  Congo 
red    medium    which    differentiates    these    two    organisms; 


NITROGEN-FIXING  ORGANISMS  OF  LEGUMES     259 

Ps.  radicicola  forms  white  colonies,  while  Bad.  tumefadens 
absorbs  the  Congo  red  and  therefore  produces  red  or  reddish 
colonies. 

B.  TEST  OF  THE  PHYSIOLOGICAL  EFFICIENCY  OF 
PS.  RADICICOLA  AND  OBSERVATION  OF  NOD- 
ULE FORMATION 

To  observe  nodule  formation  and  nitrogen  fixation,  it 
is  necessary  to  have  seeds  germinating  free  from  bacteria. 

Apparatus.  500  c.c.  of  nitrogen-free  agar;  six  sterile 
large  est  tubes  with  foot;  sterile  ordinary  test  tubes 
(the  agar  should  be  distributed  in  all  the  test  tubes  to  a 
depth  of  about  5  cm.);  sterile  Petri  dishes;  clean  slides; 
mercuric  chloride,  1  :  500;  flask  of  sterile  distilled  water; 
sterile  pipette;  seeds  of  some  leguminous  plant.  (The> 
smaller  seeds  are  better  for  this  experiment.) 

Culture.     Ps.  radidcola  (specific  strain). 

Method.  1.  Obtain  sound,  mature  pods  of  some  legume 
as  pea,  bean,  vetch,  etc. 

For  testing  the  physiological  efficiency  of  the  pure 
culture  of  Ps.  radicicola  just  previously  isolated,  use  seeds 
from  the  same  legume  as  that  from  which  this  particular 
culture  was  isolated. 

2.  Soak  the  pods  for  five  minutes  in  mercuric  chloride 
1  :  500  and  remove  the  excess  of  solution  with  sterile  cheese- 
cloth. 

3.  Tear  open  the  pods  with  flamed  forceps,  place  the 
seeds  between  folds  of  sterile  cotton,  and  put  the  cotton  in 
a  dry,  warm  place  until  the  seeds  are  dry. 

4.  Select  the  best  of  these  seeds  and  store  them  in  dry 
sterile  test  tubes  until  they  are  to  be  used. 

6.  Whether  seeds  are  procured  as  described  above,  or 
otherwise,  proceed  as  follows: 

(a)  Soak  the  leguminous  seed  in  1  :  500  mercuric  chloride 
solution  for  five  minutes. 


260 


GENERAL  MICROBIOLOGY 


(6)  Wash  off  the  disinfectant  with  sterile  distilled  water, 
handling  the  seeds  with  sterile  forceps. 


FIG.   56. — Alfalfa  Plants  from   Inoculated  and   Uninoculated    Seed. 
(Orig.  Northrup.) 


6.  Seeds  parepared  as  above   should  then  be  treated 
according  to  the  following  procedure : 

Using  the  sterile  forceps,  transfer  several  of  these  ster- 


NITROGEN-FIXING  ORGANISMS  OF  LEGUMES     261 

ilized  seeds  to  each  of  the  large  test  tubes.  Or,  place  the 
seeds  between  layers  of  moist  sterile  filter  paper  in  a  Petri 
dish  until  they  have  germinated  and  then  transfer  the  seeds 
to  the  large  test  tubes.  When  using  the  larger  seeds  use 
only  three  to  six  per  tube,  and  six  to  ten  only  of  the  smaller 
seeds  as  alfalfa,  clover,  etc. 

7.  Put  the  tubes  containing  the  ungerminated  seed  in  a 
warm  place   (30°   to  35°   C.)   until    the    seeds    germinate. 
Keep  the  germinated  seed  in  a  well-lighted  room  for  a  few 
days. 

8.  Examine  the  tubes  and  reject  all  that  are  contaminated 
with  molds  or  bacteria. 

9.  After  a  few  days,  inoculate  four  of  these  tubes  contain- 
ing growing  leguminous  plants  with  a  pure  culture  of  Ps. 
radicicola,  by  dropping  upon  the  seeds  and  surface  of  the 
agar  a  heavy  suspension  of  the  bacteria  in  sterile  water,  by 
means  of  a  sterile  pipette. 

10.  Keep  two  tubes  uninoculated  as  controls. 

Note.  To  imitate  infection  under  more  natural  conditions,  just 
before  the  seeds  are  placed  upon  the  agar,  the  agar  may  be  melted, 
cooled  to  40°  to  45°  C.  and  inoculated  with  a  loopful  of  Ps.  radicicola 
culture,  mixing  the  organisms  well  through  the  agar  with  the  needle. 
After  the  agar  has  solidified  the  sterile  seeds  may  be  then  placed  on  the 
surface  of  the  agar  as  before. 

11.  Label  the   test  tubes  and  place  in  some  location 
where  they  will    be    sufficiently  protected    from    the   sun, 
heat,   or  cold,   etc.     This  is  very  important.     A   piece   of 
cheese-cloth  thrown  over  the  tubes  will  protect  them  from 
the  sun. 

12.  In  about  a  month  examine  all  test  tubes  and  look 
for  nodules. 

13.  Record  the  presence,  number,   size,   and   shape   of 
nodules,    place   of   formation,    etc.     Show   nodule-bearing 
seedlings  to  the  instructor. 

14.  Isolate  Ps.  radicicola  from  one  of  these  nodules. 
This  completes  the  cycle. 


262  GENERAL  MICROBIOLOGY 

If  all  of  these  operations  are  successful  Koch's  postu- 
lates have  been  fulfilled.  (See  reference,  W.  J.  MacNeal, 
Exercise  1,  Animal  Diseases  and  Immunity.) 

15.  What    do    you    conclude    as    to    the    physiological 
efficiency  of  the  culture  of  Ps.  radicicola  used? 

16.  What  several  factors  might  be  responsible  for  a 
failure  of  infection?     Explain. 

Why  are  inoculated  seeds  kept  from  direct  sunlight? 
What  may  be  the  advantage  of  seed  inoculation?     How 


FIG.  57. — Ps.  radicicola,  Polar   flagella,  x!500.     Twenty-five  day  old 
culture  from  sweet  pea.     (B.  Barlow). 

do  different  methods  of  seed  inoculation  compare  as  to 
advantages  and  disadvantages? 

17.  Give  your  results  in  detail  and  draw  conclusions. 
Point  out  any  possible  practical  applications. 

REFERENCES 

MARSHALL:    Microbiology,  pp.  273-283. 

LAFAR:     Technical  Mycology,  Vol.  I,  pp.  259-271. 

LOHNIS:     Laboratory    Methods    in    Agricultural    Bacteriology,    pp. 

111-113. 
LIPMAN  and  BROWN;     Laboratory  Guide  in  Soil  Bacteriology,  pp. 

56-59,  76-77, 


CHANGE  OF  INSOLUBLE  PHOSPHATES          263 

SMITH,   E.   F.:    Bacteria  in  Relation  to  Plant  Diseases.    Vol.  II, 

pp.  96-138. 
Inoculation  with  Nodule-forming  Bacteria,  Cir.  5,  Mich.  Exp. 

Sta.,  1915. 
FRED,  E.  B.:  A  Physiological  Study  of  the  Legume  Bacteria,  Ann. 

Kept.  Va.  Polytechnic  Institute,  1911,  1912,  pp.  174-201. 

EXERCISE  10.  TO  DEMONSTRATE  THE  CHANGE  OF 
INSOLUBLE  PHOSPHATES  TO  A  SOLUBLE  FORM 
THROUGH  THE  AGENCY  OF  MICROORGANISMS 

Apparatus.  Dextrose;  di-  or  tri-calcium  phosphate; 
tubes  of  soil-extract  agar  containing  2%  dextrose;  four 
100  c.c.  Erlenmeyer  flasks. 

Culture.    Soil. 

Method.  1.  Place  0.1  gm.  of  di-  or  tri-calcium  phos- 
phate, and  60  c.c.  of  a  2%  solution  of  dextrose  in  tap  water 
in  each  flask.  Sterilize. 

2.  To  two  flasks  add  0.1  gm.  soil  each,  leaving  two  for 
controls. 

3.  Incubate  at  37°  C.,  and  after  the  fermentation  has 
continued  for  some  days,  make  plates  from  the  inoculated 
flasks  as  follows: 

4.  Sterilize  about  0.1  gram  of  di-  or  tri-calcium  phos- 
phate in  each  of  three  test  tubes. 

5.  Place  the  contents  of  each  tube  in  a  sterile  Petri  dish; 
make  loop-dilution  plates  from  flasks  in  soil  extract  agar 
containing  2%   dextrose,  being  careful  to  mix  the  phos- 
phate well  with  the  agar  in  the  dish  by  carefully  tilting. 

6.  Incubate  at  37°  C. 

7.  Note  frequently  the  appearance  of  the  plates.     The 
colonies    of    acid-producing    bacteria    developing    at    this 
temperature  dissolve  the  phosphate  and  thus  become  sur- 
rounded by  a  clear  area  similar  to  that  produced  by  lactic 
acid-producing  bacteria  on  dextrose  calcium  carbonate  agar. 

8.  Examine   the   colonies  in  a  hanging  drop  for  mor- 
phology, motility,  etc. 

9.  How  is  the  action  noted  in  7  made  use  of  practically? 


264  GENERAL  MICROBIOLOGY 

In  what  compounds  is  phosphorus  found  in  soil?  Are 
these  available  as  plant  food?  What  are  the  functions  of 
bacteria  in  this  connection? 

What  relation  has  phosphorus  to  decay  and  nitrogen 
fixation? 

10.  Give  results  and  any  conclusions  in  detail.  Point 
out  any  possible  practical  applications. 

REFERENCES 

MARSHALL:     Microbiology,  pp.  287-288. 

LOHNIS:     Laboratory  Methods  in  Agricultural  Bacteriology,  pp.  115- 

116. 
SACKETT,  PATTEN  and  BROWN:    The  Solvent  Action  of  Soil  Bacteria 

Upon  Insoluble  Phosphates,  etc.,  Spec.  Bui.  43,  Mich.  Exp.  Sta., 

1908. 

DAIRY  MICROBIOLOGY 

EXERCISE  1.  A  COMPARATIVE  STUDY~OF  THE  NUM- 
BER AND  TYPES  OF  MICROORGANISMS  AND 
OTHER  CELLS  IN  MILK 

A.    PLATING  METHOD 

Apparatus.  Sterile  Petri  dishes;  sterile  1  c.c.  and  10 
c.c.  pipettes;  tubes  of  sterile  litmus  lactose  agar;  90  c.c. 
and  99  c.c.  dilution  flasks;  tubes  of  sterile  litmus  milk. 

Culture.     Fresh  milk  from  any  source  desired. 

Method.  1.  Shake  the  milk  vigorously  one  hundred 
times  to  obtain  a  homogeneous  sample,  and  plate  in  dilu- 
tions, 1-100,  1-10;000  and  1-1,000,000  in  litmus  lactose 
agar. 

2.  Incubate  the  plates  inverted  at  room  temperature 
for  five  days. 

3.  Count  at  the  end  of  this  time,  estimate  the  average 
number  of  bacteria  per  cubic  centimeter  and  approximate 
the  numbers  of  the  different  types  of  colonies. 

4.  Are   acid   colonies   present?     Chromogenic   colonies? 
B.  subtilis  or  B.  mycoides? 


TYPES  OF    MICROORGANISMS  IN  MILK 


265 


What  do  the  types  signify? 

5.  Isolate  the  different  types  in  litmus  milk  and  note 
their  action.  To  which  group  of  microorganisms  does 
each  type  belong?  (See  Marshall's  Microbiology,  pp. 
306-313.)  Suggest  from  what  source  each  type  may  come. 

B.    MICROSCOPIC  METHOD 

Apparatus.  Special  capillary  pipettes  graduated  to 
deliver  exactly  0.01  c.c.;  clean  glass  slides; 
three  staining  jars;  xylol;  alcohol,  95%; 
Loeffler's  alkaline  methylen  blue;  stage 
micrometer;  eyepiece  micrometer  for 
counting  objects  in  microscopic  field;  stiff 
straight  needle. 

Culture:  Fresh  milk — same  as  used 
in  A. 

Method.  1.  Draw  with  ink  a  figure 
the  size  and  shape  of  an  ordinary  micro- 
scopic slide  and  on  either  side  and  equi- 
distant from  the  center  draw  a  square 
whose  area  is  one  square  centimeter, 
making  the  homologous  sides  of  all  figures 
parallel. 

2.  Place  a   clean  glass   slide   on  the 
figure. 

3.  With   the    capillary   pipette,   drop 
over   the    center   of  one  of  the   smaller 

figures   exactly  0.01  c.c.   of  milk  directly  FIG.  58.— Capillary 
from  the  well-shaped  sample  and  with  a      plPette  usea  in 
stiff  straight  needle  spread   this   drop  of 
milk  exactly  over  the   area  (one   square 
centimeter)  covered  by  this  figure. 

4.  Make   a    duplicate    smear,  placing 
the  drop  of  milk  containing  0.01  c.c.  on 
the  slide  over  the  remaining  small  square. 

5.  These  smears  may  be  dried  by  the  use  of  gentle 


the  Microscopic 
Method  for 
Counting  B  a  c  - 
teria  in  Milk. 
Note  the  straight 
narrow  bore  and 
the  square  tip. 


266  GENERAL  MICROBIOLOGY 

heat  (e.g.,  level  wooden  surface  over  a  steam  radiator). 
Do  not  allow  the  smears  to  become  too  hot,  as  this  causes 
them  to  check,  making  satisfactory  staining  impossible. 

6.  As  soon  as  dry,  place  the  slides  in  a  staining  jar 
containing  xylol  for  a  short  time  to  remove  the  fat. 

7.  Remove  the  slide  from  the  xylol,  absorb  the  surplus 
xylol   about   the   edges   with   filter   paper  and  allow  it  to 
dry. 

8.  Fix  the  film  to  the  slide  by  immersing  in  95%  alcohol. 

9.  Stain  immediately  by  flooding  the  smears  with  Loef- 
fler's  methylen  blue  for  two  or  three  minutes. 

10.  Decolorize  to  a  light  blue  in  95%  alcohol. 

11.  In  counting,  use  the  oil  immersion  objective.     Place 
the  draw  tube  at  some  convenient  mark  so  that  an  even 
number  of  fields  of  the  microscope  covers  one  square  centi- 
meter. 

To  do  this,  determine  the  radius  of  the  -microscopic 
field  in  millimeters  with  the  stage  micrometer  and  calculate 
its  area  by  the  formula  irR2.  (7r  =  3.1416.) 

Then  if  z  =  the  area  of  the  smear  in  square  millimeters 
and  if  0.01  c.c.  of  milk  is  used, 


y  —  the  factor  necessary  to  transform  the  number  of  bacteria 
found  in  one  field  of  the  microscope  into  terms  of  bacteria 
per  cubic  centimeter. 

TO  simplify  the  calculation,  place  the  draw  tube  so  that 
y  consists  of  as  many  ciphers  as  possible.  Convenient 
factors  will  be  obtained  if  the  length  of  R  be  0.101  mm. 
or  0.08  mm. 

Let  z  thousand  equal  the  number  of  fields  of  the  micro- 
scope in  one  square  centimeter.  Since  0.01  c.c.  of  milk  was 
taken  then  each  bacterium  seen  in  one  field  represents  lOOXz 
thousand  or  z  hundred  thousand  bacteria  per  cubic  centi- 
meter. 


TYPES  OF  MICROORGANISMS  IN  MILK 


267 


12.  For  careful  quantitative  work  it  is  necessary  to  count 
one  hundred  fields  for  each  sample,  i.e.,  fifty  fields  per  square. 
If  n  =  the  number  of  fields  counted  and  m  =  the  total  num- 
ber of  bacteria  found,  the  number  of  bacteria  per  cubic 
centimeter  is  calculated  by  the  following  formula: 


z  hundred  thousand 


centimeter  of  milk. 


=  number    of     bacteria    per    cubic 


In  comparatively  fresh  milk  where  the  bacteria  are  few, 
count  the  whole  microscopic  field. 

An  eye-piece  micrometer  having  a  large  square  ruled 
into  smaller  squares  is  recommended  where  large  numbers 
of  bacteria  are  present.  The  area  of  the  large  square  is 
different  from  that  of  the  whole  microscope  field  and  con- 
sequently the  factor  necessary  for  computation  is  different. 
This  factor  can  be  determined  by  modification  of  the  formula 
given  in  11. 

13.  Draw  a  typical  smear  from  different  samples  of  milk. 
Indicate  the  kinds  of  cells  and  the  number  found,  also  the 
quality  of  the  milk. 


Quality  of  milk. 

Bacteria 
per  field. 

No.  per  c.c. 

Tissue 
cells. 

Cell  count. 

Good  
Fair  

None 
5 

2,000,000 

2 

1 

800,000  per  c.c. 
400,000  per  c.o, 

Souring  normally 
Poor  

200 
250 

80,000,000 
100,000,000 

1 

7 

400,000  per  c.c. 
2,800,000  per  c.c. 

14.  What  types  of  bacteria  are  found  microscopically? 
How  do  these  compare  with  those  found  on  plates  as  to 
types  and  numbers? 

What  are  the  advantages  and  disadvantages  of  the  plat- 
ing method?  Of  the  microscopic  method?  For  what  type 
of  work  is  each  best  adapted?  What  other  microscopic 


268  GENERAL  MICROBIOLOGY 

methods  have  been  employed  as  a  rapid  means  of  setting 
bacteriological  milk  standards? 

Of  what  value  are  bacteriological  milk  standards  and 
analyses? 

15.  Give  your  results  in  detail  and  point  out  any  prac- 
tical applications. 

REFERENCES 

BREW,  JAMES  D.:   A  Comparison  of  the  Microscopical  Method  and 

Plate  Method  of  Counting  Bacteria  in  Milk.'     Bui.  373,  N.  Y. 

Agr.  Expt.  Sta.,  Geneva,  Feb.,  1914. 
MARSHALL:     Microbiology,  pp.  293-296,  331-333. 
LOHNIS:     Laboratory    Methods    in    Agricultural    Bacteriology,    pp. 

62-65. 
SAVAGE:     The  Bacteriological  Examination  of  Food  and  Water  (1914), 

p.  85-89,  92,  95-99. 

WARD:     Pure  Milk  and  the  Public  Health  (1909),  pp.  126-128. 
ERNST:     Milk  Hygiene;    translated  by  Mohler  and  Eichhorn  (1914), 

pp.  24-31. 
FROST,  W.  D.:     A  Microscopic  Test  for  Pasteurized  Milk.     Jour.  Am. 

Med.  Assn.     Vol.  LXIV,  No.  10,  p.  821  (1915). 

EXERCISE  2.     THE  DETERMINATION  OF  THE  BACTE- 
RIAL CONTENT  OF  MILK  IN  THE  UDDER 

Apparatus.  Several  large  sterile  test  tubes;  four 
sterile  Petri  dishes;  99  c.c.  dilution  flasks;  sterile  1  c.c. 
pipettes;  tubes  of  sterile  litmus  milk;  four  tubes  litmus 
lactose  agar. 

Method.  1.  Wash  off  the  end  of  the  teat  very  care- 
fully with  a  solution  of  mercuric  chloride,  1  :  1000;  allow 
it  to  dry  till  the  surplus  solution  has  disappeared  and 
only  sufficient  moisture  remains  to  make  the  cells  and  any 
dirt  adherent. 

2.  Secure  a  sterile  cotton-plugged  test  tube,  remove 
the  cotton  pliig  with  the  little  finger  and,  while  holding  the 
mouth  of  the  tube  as  near  the  end  of  the  sterilized  teat  as 
possible  and  inclining  the  tube  toward  the  horizontal  posi- 
tion as  far  as  feasible,  milk  the  tube  half-full. 


BACTERIAL  CONTENT  OF  MILK  IN  THE  UDDER   269 

By  this  method  obtain  from  the  same  teat: 

a.  One  sample  of  the  fore  milk, 

b.  One  sample  of  the  middle  milk; 

c.  One  sample  of  the  strippings; 

Note.  For  investigational  purposes  it  may  be  better  to  employ 
a  sterile  milking  tube  adjusted  to  a  sterile  flask.  This  may  easily  be 
prepared.  This  method,  however,  is  not  recommended  for  student 
work. 

3.  Secure  one  sample  from  the  pail,  gathered  from  the 
same  cow  at  the  same  milking. 

4.  Plate  each  sample  on  litmus  lactose  agar,  using  the 
following  dilutions  and  amounts: 

1  c.c.  of  2a  diluted  1  :  100  for  the  plate. 
1  c.c.  of  26  diluted  1  :  10  for  the  plate. 
1  c.c.  of  2c  diluted  1  :  10  for  the  plate. 
1  c.c.  of  3  diluted  1  :  100  for  the  plate. 

5.  Place  the  plates  at  a  temperature  of  21°  C.  for  seven 
days. 

6.  Count   the   number   of  colonies  in   each   plate   and 
record  the  average  number  in  1  c.c.  of  milk  in  each  case. 
Explain  any  variation  in  counts. 

7.  Compare  the  colonies  of  plates  2a,  26,  and  2c  with 
3.     What  types  predominate? 

8.  Estimate,  so  far  as  possible,  the  number  of  colonies 
of  each  type,  and  compare  the  relative  numbers  of  each 
species  in  the  different  plates. 

9.  Isolate  the  species  in  milk  tubes  to  study  their  action 
upon  milk.     To  what  group  of  microorganisms  found  in 
milk  does  each  of  the  species  isolated  belong?     How  do  you 
account  for  the  presence  of  these  particular  species? 

10.  In  which  sample  would  you  expect  to  find  the  greatest 
number  of  microorganisms?     Why? 

Why  should  all  samples  be  taken  from  the  same  quarter 
of  the  udder? 

How  do  bacteria  ordinarily  gain  entrance  to  the  udder? 


270  GENERAL  MICROBIOLOGY 

By  what  means  may  bacteria  cause  infection  of  the 
udder?  _ 

What  is  the  significance  of  bacteria  in  the  udder?  As 
to  numbers  and  types? 

11.  Give  your  results  in  full  and  point  out  any  conclu- 
sions and  any  practical  applications  possible. 

REFERENCES 

MARSHALL:     Microbiology,  pp.  297-299,  306-313. 
WARD:    Pure  Milk  and  the  Public  Health  (1909),  pp.  1-7,  69,  86-88. 
ROSENAU:    The  Milk  Question  (1912),  pp.  71-74. 
RUSSELL   and   HASTINGS:    Outlines   of   Dairy   Bacteriology    (1910), 
pp.  30-35,  75. 

EXERCISE    3.     TO    ILLUSTRATE    EXTRANEOUS    CON- 
TAMINATION 

Apparatus.  Forceps;  scalpel  or  spatula;1  seventeen 
sterile  Petri  dishes;  three  10  c.c.  dilution  flasks  (for  A  and  B) ; 
fifteen  sterile  1  c.c.  pipettes;  fifteen  tubes  of  sterile  litmus 
lactose  agar;  soap;  ordinary  towel;  two  1  qt.  sterile  basins; 
one  milk  pail;  two  1  liter  flasks  each  containing  500  c.c. 
sterile  salt  solution;  one  deep  Petri  dish;  sterile  glass  rod. 

A.    SCALES  FROM  COW'S  SKIN  (DEAD  EPITHELIAL 

CELLS) 

Method.  1.  With  a  sterile  spatula  scrape  from  the 
skin  of  a  cow's  udder  some  scales,  such  as  usually  fall  into 
the  milk,  into  a  sterile  Petri  dish. 

2.  Transfer  by  means  of  the  sterile  spatula  some  of  these 
to  a  75  c.c.  Erlenmeyer  flask  containing  10  c.c.  of  sterile 
physiological  salt  solution. 

3.  Shake  thoroughly,  then  plate  1  c.c.  of  this  suspension 
in  litmus  lactose  agar, 


TO  ILLUSTRATE  EXTRANEOUS  CONTAMINATION     271 


B.    HAIRS  FROM  COW 

Method.  1.  Select  two  hairs  from  the  back  of  the  cow 
where  the  usual  or  natural  clean- 
liness exists  and  two  from  the 
hip  stained  with  manure.  By 
means  of  sterile  forceps  place 
them  in  sterile  Petri  dishes. 

2.  One  of  each  kind,  that  is, 
one  from  the  back  and  one  from 
the   hip,  place   in    10  c.c.   of  a 
sterile  salt  solution,  as  under  A. 

3.  Shake     thoroughly,    then 
plate  1  c.c.  of  this  suspension  in 
litmus  lactose  agar. 

4.  Embed  each   of   the  two 
remaining    hairs    in   the  litmus 
lactose    agar   after   pouring  the 

liquefied  agar  into  a  sterile  Petri  dish.  These  hairs  should 
be  placed  in  the  agar  just  before  solidifying  by  means  of 
sterile  forceps. 


FIG.  59. — Bad.  bulgaricum 
colony,  x75.  (Orig.  Nor- 
thrup.) 


C.    OTHER  SUBSTANCES 

Method.  Study  straw,  hay,  dung,  etc.,  in  a  similar 
manner.  Instead  of  using  dilution  flasks  containing  10  c.c. 
it  will  be  more  desirable  to  use  100  c.c. 

In  the  case  of  dung,  a  particle  smaller  than  the  head 
of  a  pin  should  be  added  to  100  c.c.  for  suspension,  and 
in  case  of  straw  and  hay  very  small  segments  unless  they 
are  very  clean. 

Note.  A  sufficient  number  of  such  substances  should  be  studied 
to  familiarize  the  student  with  the  amount  of  contamination  which 
may  take  place  from  these  sources. 


272  GENERAL  MICROBIOLOGY 


D.     HANDS 

Method.  1.  Wash  the  hands  in  the  ordinary  manner, 
rinse  them  thoroughly,  then  wipe  with  an  ordinary  towel. 

2.  After  this  has  been  done,  put  500  c.c.  of  sterile  water 
in  a  sterile  dish,  and  rub  the  hands  thoroughly  with  this 
water. 

3.  Plate  1  c.c.  of  this  water  in  litmus  lactose  agar. 

4.  Again  rub  the  hands,  before  they  have  been  washed 
and  after  working  for  some  time,  in  500  c.c.  of  sterile  water 
placed  in  a  sterile  dish. 

6.  Plate  1  c.c.  of  this  water  in  litmus  lactose  agar. 
6.  Compare  the  numbers  (using  1  c.c.  as  the  unit)  and 
kinds  of  bacteria  in  the  two  plates. 

E.     PAILS 

Method.  1.  Add  to  a  milk  pail  washed  in  the  usual 
manner,  500. c.c.  of  sterile  salt  solution,  and  plate  in  litmus 
lactose  agar,  1  c.c.  of  this  suspension  after  it  has  been 
moved  over  the  inner  surface  of  the  pail. 

2.  Repeat  by  using  a  milk  pail  heated  in  steam  for  ten 
minutes,  or  cleansed  with  boiling  water. 

3.  This  same  process  may  be  repeated  using  milk  bottles, 
milk  cans,  etc. 

F.    AIR 

Method.  1.  Determine  qualitatively  the  microflora 
of  the  air  of  the  stable  before  feeding  or  bedding  or  before 
any  disturbing,  and  after  feeding  or  bedding  or  after  any 
disturbing,  by  the  following  methods : 

2.  Pour  the  liquefied  litmus  lactose  agar  into  several 
Petri  dishes,   and  expose  the  poured   plates  for  different 
lengths  of  time. 

3.  Expose  10  c.c.  of  sterile  0.6%  salt  solution  in  a  deep 
Petri  dish  5  c.c.  deep  and  9  c.c.  in  diameter.     Try  to  disin- 


AMOUNT  AND   KIND  OF  DIRT  IN  MILK  273 

tegrate  the  dust   particles   by  stirring  with  a  sterile  glass 
rod  and  agitating.     Plate  1  c.c.  in  litmus  lactose  agar. 

4.  Quantitative  studies  of  barn  air  under  various  con- 
ditions may  be  made  according  to  Exercise  1,  Air  Micro- 
biology. 

5.  What   advantage  has  litmus  lactose  agar  over  ordi- 
nary agar  in  this  exercise? 

What  types  of  organisms  are  met  most  frequently  under 
A,  B,  C,  D,  E  and  F?  How  may  this  occurrence  be  ac- 
counted for? 

Which  sources  furnish  the  greatest  number  of  organisms? 
From  which  sources  are  the  greatest  number  of  micro- 
organisms most  likely  to  enter  milk?  The  most  undesirable 
types?  Explain  in  each  case. 

What  sources  of  milk  contamination  have  not  been  dis- 
cussed under  this  exercise?  Of  what  importance  is  each? 
What  is  the  simplest  method  in  each  case  of  preventing 
contamination  from  the  various  sources  mentioned  above? 

6.  Give  your  results  in  full  and  draw  any  conclusions 
and  make  any  practical  applications  possible. 

REFERENCES 

SAVAGE:     The  Bacteriological  Examination  of  Food  and  Water  (1914), 

pp.  90-91. 
ERNST:     Milk  Hygiene,  transl.  by  Mohler  and  Eichhorn  (1913),  pp. 

67-102,  125-131,  234-235. 

JENSEN:     Milk  Hygiene,  transl.  by  Pearson  (1907),  pp.  70-82,  86-127. 
MARSHALL:     Microbiology,  pp.  300-306. 

EXERCISE  4.  TO  INVESTIGATE  THE  AMOUNT  AND 
KIND  OF  DIRT  IN  MILK  AND  ITS  RELATION  TO  THE 
MICROBIAL  CONTENT  OF  THE  MILK 

Apparatus.  Six  sterile  1  c.c.  pipettes;  99  c.c.  dilution 
flasks;  six  tubes  sterile  litmus  lactose  agar;  six  sterile 
Petri  dishes;  sedimentation  tubes,  10  c.c.  capacity;  balance; 
centrifuge;  clean  slides;  methylen  blue,  aqueous-alcoholic; 
physiological  salt  solution;  pneumatic  or  other  type  of 


274  GENERAL  MICROBIOLOGY 

sediment  tester;  cotton  disks  for  sediment  tester;  clean 
empty  milk  bottle;  one  pint  bottled  milk  from  each  of 
several  miscellaneous  sources. 

Note.     The  same  sample  of  milk  must  be  used  for  A,  B  and  C. 
Proceed  with  tests  in  the  order  given. 

A.     DETERMINATION  OF  MICROBIAL  CONTENT 
OF  MILK 

Method.     1.  Shake  the  sample  in  the  bottle  vigorously. 

2.  Plate  the  dilutions  1  :  100,1  :  10,000  and  1  :  1,000,000 
in  litmus  lactose  agar. 

3.  Place  the  plates  at  25°  C.,  and  proceed  with  the 
microscopic  sediment  test. 

4.  Count  the  plates  at  the  end  of  five  days  and  estimate 
the  number  of  bacteria  per  cubic  centimeter  and  the  relative 
proportion  of  acid  to  other  types  of  colonies. 

5.  Determine  the  morphology  of  the  organisms  making 
up  the  colonies  of  each  type  and  compare  with  the  findings 
in  the  microscopical  sediment  test. 

6.  Are  all  organisms  present  microscopically?     Explain 
your  results  and  draw  conclusions. 

B.     MICROSCOPIC  SEDIMENT  TEST 

Method.     1.  Mix  the  milk  well  and  warm  about  30  c.c. 
to  60°  to  70°  C. 

2.  Place  10  c.c.  of  this  well-mixed,  warmed  milk  into 
each  of  two  sedimentation  tubes. 

3.  Place  one  tube  on  each  of  the  scale  pans  and  balance 
by  adding  more  milk  to  the  lighter  tube.     The  tubes  must 
be  equal  in  weight  or  they  will  throw  the  centrifuge  "  off 
center." 

4.  Centrifuge  in  a  machine  designed  for  this  purpose  for 
five  minutes,  till  a  more  or  less  considerable  compact  sedi- 
ment separates  out. 

6.  Pour  or  pipette  off  the  milk  above  the  sediment. 


AMOUNT  AND  KIND   OF  DIRT  IN  MILK 


275 


6.  Fill  the  tubes  with  physiological  salt  solution  and  mix 
the  sediment  well  throughout  the  dilution  fluid  with  a  plat- 
inum needle. 

7.  Balance  the  tubes  and  centrifuge  again. 

8.  Pour  or  pipette  off  the  physiological  salt  solution. 

9.  With  a  small  platinum  loop,  obtain  a  small  amount 
of  the  sediment  and  make  a  smear  on  a  clean  slide. 

10.  Stain  with  aqueous-alcoholic  methylen  blue. 


FIG.  60. — Wizard  Sediment  Tester  for  Milk. 

11.  Determine  the  proportions  of  bacteria  and  leuco- 
cytes in  ten  fields.  Also  note  the  presence  of  bacteria  in 
clumps  and  foreign  matter. 

The  presence  of  many  leucocytes  and  streptococci  asso- 
ciated together  is  generally  indicative  of  an  inflamed  con- 
dition of  the  udder,  as  in  mastitis  (garget).  On  the  other 
hand,  sometimes  the  milk  from  normal  udders  may  show  a 
considerable  quantity  of  leucocytes  in  the  sediment. 


276  GENERAL  MICROBIOLOGY 


C.     MACROSCOPIC  SEDIMENT  TEST 

Method.  1.  Put  a  cotton  disk  in  place  in  the  pneu- 
matic sediment  tester,  heat  the  sediment  tester  and  clean 
empty  milk  bottle  in  steam  thirty  minutes,  and  allow  to 
cool. 

2.  Attach  the  sediment  tester  to  the  top  of  the  milk 
bottle   containing   the   sample   of  milk,   using    "  aseptic " 
precautions,  and  invert  the  whole  apparatus  over  the  mouth 
of  the  sterile  empty  milk  bottle. 

3.  Pump  the  contents  of  the  upper  bottle  into  the  lower 
bottle  by  means  of  the  rubber  bulb.     The  milk  is  forced 
through  the  cotton  disk  and  leaves  its  larger  particles  of 
insoluble  dirt  on  the  cotton. 

4.  Note  the  quality  of  the  milk  tested  by  this  method. 
Is  there   any  interrelationship   between  microscopic   sedi- 
ment test,  and  the  macroscopic  sediment  test?  ,  , 

5.  What  does  the  presence  of  visible  dirt  on  the  cotton 
indicate?     Is  this  sediment  te^t  an  argument  for  straining 
milk  before  it  goes  to  the  consumer?     Is  it  an  argument 
for  running  milk  through  a  milk  clarifier  before  putting  it 
on  the  market? 

6.  Immediately  after  straining,  plate  the  milk   in  lit- 
mus lactose  agar,  using   dilutions  1  :  100,   1  :  10,000   and 
1  :  1,000,000  as  before. 

7.  Incubate,  the  plates  for  five  days  at  25°  C.  and  count, 
estimating  total  average  number  and  proportions  of  types 
as  in  A. 

8.  Compare  the  counts  with  those  of  A,  also  the  propor- 
tions of  the  various  types. 

Note.  This  method  was  formerly  used  for  obtaining  an  estimate 
microscopically  of  the  numbers  of  bacteria  in  milk.  It  presents 
difficulties,  however,  which  lead  to  many  technical  errors  and  there- 
fore it  cannot  be  relied  upon  to  give  uniform  results.  The  method  is 
valuable,  however,  for  determining  something  of  the  sanitary  quality 
of  the  milk, 


AMOUNT  AND  KIND  OF  DIRT  IN  MILK  277 


FIG.  61. — Cotton  Disks  Prepared  by  the  Use  of  the  Wizard  Sediment 
Tester,     (Circ.  41,  Wise.  Expt.  Sta.) 


278  GENERAL  MICROBIOLOGY 

Did  straining  have  any  effect  on  the  numbers  of  organisms 
present  in  the  milk?  What  effect  may  it  have?  Is  this 
beneficial  to  the  milk  as  a  commercial  product? 

9.  In   what  way  may  the  microscopic   sediment   test 
explain  the  results  obtained  by  plating  milk  before  and  after 
straining? 

10.  Make,  stain  and   examine  smears   from  the  upper 
surface  of  the  cotton  disk.     What  is  the  nature  microscopic- 
ally of  the  material  retained  by  the  cotton?     How  does  this 
smear  compare  qualitatively  with  that  from  the  centri- 
fuged  sample? 

11.  What  is  the  nature  of  the  dirt  ordinarily  found  in 
milk?     How  may  its  presence  be  eliminated? 

12.  Give  all  results  in  full   and  draw  any  conclusions 
permissible,    Point  out  any  practical  applications. 

REFERENCES 

LOHNIS:     Laboratory  Methods  in  Agricultural  Bacteriology,  pp.  63-65. 
JENSEN:     Milk  Hygiene,  transl.  by  Pearson  (1907),  pp.  126-127. 
MARSHALL:     Microbiology,  pp.  326-327. 
ROSENAU:    The  Milk  Question  (1912),  pp.  55-88. 
ERNST:     Milk  Hygiene,  transl.  by  Mohler  and  Eichhorn  (1914),  p. 
182. 


EXERCISE  5.  TO  DETERMINE  THE  INFLUENCE  OF 
TEMPERATURE  UPON  THE  KEEPING  QUALITY  OF 
MILK;  PITRE  MILK  COMPARED  WITH  MARKET 
MILK 

One  of  the  most  important  considerations  in  the  pro- 
duction of  milk,  either  for  factory  use  or  for  town  or  city 
supply,  is  the  temperature  at  which  the  milk  is  maintained. 
The  beneficial  effects  of  scrupulous  cleanliness  in  the  pro- 
duction of  milk  will  be  largely  counteracted  unless  the  milk 
is  cooled  immediately  after  drawn  and  maintained  at  a 
temperature  too  low  for  development  of  the  bacteria 
present. 


PUKE  MILK  COMPARED  WITH  MARKET  MILK     279 

Apparatus.  Three  sterile  1  liter  Erlenmeyer  flasks; 
one  sterile  2  liter  Erlenmeyer  flask;  twenty-four  sterile 
Petri  dishes;  sterile  10  c.c.  pipettes;  sterile  1  c.c.  pipette 
graduated  to  0.1  c.c.;  twenty-four  tubes  of  litmus  lactose 
agar;  90  c.c.  and  99  c.c.  dilution  flasks;  ice  and  salt  for  pre- 
paring freezing  mixture;  fresh  milk  and  bottled  milk. 

Method.  1.  In  a  sterile  2  liter  Erlenmeyer  flask  place 
about  1 J  liters  of  milk  from  a  can  of  milk  immediately  after 
it  has  been  filled  by  the  milkers. 

Note.  This  exercise  is  to  be  repeated,  for  purposes  of  comparison, 
using  three  pint  bottles  of  milk  all  obtained  at  one  time  from  the 
same  milkman.  In  this  case  the  first  plating  is  to  be  made  from 
each  separate  bottle. 

2.  Record   the   temperature.     Plate    from   the   sample 
immediately  in  litmus  lactose  agar,   making  dilutions  of 
1  :  100  and  1  :  500.     Determine  the  acidity  of  the  sample, 
using  a  sterile  5  c.c.  pipette  to  obtain  the  sample. 

Note.  Portions  for  acidity  determination  and  plating  should  be 
removed  with  sterile  pipettes  in  all  instances. 

3.  Transfer  the    sample    "  aseptically  "  into  the  three 
1  liter  flasks,  placing  an  equal  portion  as  nearly  as  possible 
in  each  flask.     Label  the  flasks  A,  B,  C. 

4.  Cool  flask  A  in  a  freezing  mixture  to  10°  C.,  and  set 
away  in  refrigerator  to  maintain  the  low  temperature. 

5.  Cool  flask  B  in  a  freezing  mixture  to  10°  C.,  then  place 
it  at  a  constant  temperature  of  21°  C. 

6.  Place  flask  C  at  a  constant  temperature  of  21°  C. 

7.  At  the  end  of  twenty-four  hours  determine  and  record 
the  acidity   of  each  of  the  three  portions  of  the  original 
sample. 

8.  Plate   in    litmus   lactose    agar,   using  the   following 
dilutions : 

Flask  A,  1  :  100  and  1  :  1,000. 

Flasks  B  and  C,  1  ;  10,000  and  1  ;  1,000,000. 


280  GENERAL  MICROBIOLOGY 

9.  At  the  end  of  another  twenty-four  hours  repeat  the 
titrations  and  platings  with  all  flasks,  using  the  following 
dilutions : 

Flask  A,  1  :  10,000  and  1  :  1,000,000. 

Flasks  B  and  C,  1  :  1,000,000  and  1  :  100,000,000. 

10.  At  the  end  of  five  days  determine  and  record  the 
acidity  of  the  milk  in  all  flasks.     Plate  from  flask  A  only, 
using  dilutions  of  1  :  1,000,000  and  1  :  100,000,000. 

11.  All  plates  should  be  held  at  21°  C.  for  a  period  of 
five  days  before  counting. 

12.  Compare  the  relative  kinds  and  numbers  of  colonies 
in  plates  from  the  three  flasks.    Note  also  the  time  of  curd- 
ing and  the  nature  of  the  curd  formed  in  each  case. 

13.  Compile  the  results  of  the  investigation  in  tabulated 
form.     Plot  bacterial  count  and  acidity  curves. 

14.  From  the  results  obtained,  what  conclusions  would 
you  draw  as  to  the  influence  of  cooling  upon  the  keeping 
quality  of  milk? 

How  does  the  age  and  original  quality  of  the  milk  effect 
its  keeping  qualities  when  subjected  to  different  temperature 
conditions? 

How  dpes  cooling  milk  and  keeping  it  cool  compare  with 
merely  cooling  and  then  allowing  the  milk  to  acquire  the 
temperature  of  the  room?  What  is  the  explanation  of  the 
action  occurring? 

What  is  the  purpose  of  cooling  the  milk  as  soon  as  it 
comes  from  the  cow?  What  different  methods  are  used? 
What  are  some  of  the  disadvantages  of  the  different  methods 
used  for  cooling? 

What  bacterial  action  takes  place  in  the  refrigerator 
milk?  Is  the  germicidal  action  of  milk  sufficiently  important 
to  recommend  a  change  in  the  general  practice  of  cooling 
milk? 

15.  Give    your   results   in    detail    and    point    out    any 
practical  applications  or  conclusions. 


PASTEURIZATION  OF  MILK  OR  CREAM  281 

REFERENCES 

WARD:     Pure  Milk  and  the  Public  Health  (1909),  pp.  15-16,  24-25, 

37,  121. 

RUSSELL  and  HASTINGS:     Outlines  of  Dairy  Bacteriology,  pp.  54-56. 
ERNST:     Milk  Hygiene,  transl.  by  Mohler  and  Eichhorn  (1914),  pp. 

148-149,  156. 
MARSHALL:     Microbiology,  pp.  318-319. 


EXERCISE  6.x   A  STUDY  OF  THE  PASTEURIZATION  OF 
MILK  OR  CREAM  BY  LABORATORY  METHODS 

Apparatus.  Water  bath;  test-tube  rack  of  metal  to 
fit  water  bath;  sterile,  large  tubes  selected  for  uniformity 
in  diameter  (2  cm.);  sterile  Petri  dishes;  sterile  1  c.c.  pi- 
pettes, graduated  to  0.1  c.c.;  sterile  litmus  lactose  agar 
tubes. 

Method.  1.  Secure  milk  or  cream,  about  125  c.c.  to 
be  used  for  tubing  and  pasteurizing. 

Note.     If  time  permits,  it  is  desirable  to  test  pasteurization  upon: 

a.  Fresh  milk  or  cream. 

b.  Milk  or  cream  which  has  stood  for  twenty-four  hours  but  is 
still  sweet. 

c.  Milk  or  cream  which  has  reached  an  acidity  of  about  22°. 

d.  Milk  or  cream    from   different  sources,  supposedly  having  dif- 
ferent bacterial  contents. 

2.  Tube  the  sample  or  samples  of  milk  or  cream,  pour- 
ing 10  c.c.  into  each  tube,  filling  fifteen  tubes  for  each  sample. 

Note.     Only  one  sample  should  be  pasteurized  at  a  time. 

3.  Prepare  one  tube  from  each  sample  of  milk  or  cream 
for  the  introduction  of  the  thermometer.     By  so  doing, 
the  conditions  practically  identical,  the  temperature  will 
be  easily  read  and  controlled. 

4.  After  the  tubes  are  prepared  mark  tubes  in  duplicate 
as  follows:    50°,  60°,  70°,  80°,  90°  and  100°,  leaving  two 
unmarked  as  controls. 

5.  Place  them  in  the  rack  so  that  the  marks  on  the  tubes 


282  GENERAL  MICROBIOLOGY 

may  be  easily  recognized,  and  insert  the  rack  in  the  water- 
bath. 

6.  Pour  water  into  the  water-bath    until  the  height  of 
the  water  corresponds  to  the  height  of  the  milk  in  the 
tubes. 

7.  Put  aside  two  tubes  of  milk  or  cream  from  each 
sample,  one  to  be  employed  for  comparative  check-obser- 
vation, and  the  other  for  check-plating  against  those  which 
will  be  subjected  to  pasteurization. 

8.  Apply  heat  to  the  water-bath. 

9.  At  50°,  60°,  70°,  80°,  90°  and  100°  C.,  remove  two 
tubes  of  each  sample  of  milk  or  cream  undergoing  pasteuri- 
zation and  place  in  cold  water. 

10.  Employ  one  of  the  tubes  thus  removed  for  plating 
and  the  other  place  at  a  temperature  of  25°  to  28°  C.  along 
with  the  previous  check-observation  tube  (7). 

11.  Make  two  plates  in  litmus  lactose  agar  from  the  tube 
held  for  check-plating  (7)  and  from  one  of  the  two  tubes 
removed   at   each  of   the  temperatures  designated  above. 
The  remaining  tube  is  to  be  left  undisturbed  and  placed 
at  25°  C.,  to  observe  macroscopical  changes. 

Dilutions  for  plating: 

Fresh  milk,  unpasteurized,  1  :  10  and  1  :  100. 

Milk  twenty-four  hours  old,  but  sweet,  unpasteurized, 
1  :  10,000  and  I  :  1,000,000. 

Milk  with  an  acidity  of  22°,  unpasteurized,  1  :  100,000 
and  1  :  10,000,000. 

Milk  pasteurized  at  50°  C.  (fresh)  1  :  10  and  1  :  100. 

Milk  pasteurized  at  50°  C.  (old)  1  :  10,000  and 
1  :  1,000,000. 

Milk  pasteurized  at  60°  C.,  1  :  10  and  1  :  100. 

Milk  pasteurized  above  60°  C.,  1  :  10. 

12.  Keep  the  plates  at  25°  C.  for  seven  days,  counting 
colonies  at  the  end  of  this  time. 

13.  Determine    the    character    of    the    microorganisms 
left  after  pasteurization  with  those  before  pasteurization 


PASTEURIZATION   OF  MILK  OK  CREAM  283 

as  to  the  relative  number  of  each  kind,  to  the  fermentation 
of  milk  or  cream,  to  spore  formation,  and  to  resistance. 
Which  microorganisms  have  succumbed  to  pasteurization 
at  different  temperatures  and  which  were  able  to  withstand 
it? 

14.  Record  the  results  obtained  from  the  study  of  plates 
and  cultures  made  from  colonies. 

15.  Record  your  observations  from  day  to  day  of  macro- 
scopical  changes  in  the  pasteurized  and  unpasteurized  con- 
trol  tubes.     Does   pasteurization   destroy   organisms   that 
are  favorable,  or  detrimental  to  the  milk?     What  influence 
does    pasteurization    have    upon    the    keeping    quality    of 
milk?   . 

16.  What  influence  do  the  following  factors  have  upon 
the  efficiency  of  pasteurization:    the  age  of  milk?    acidity? 
degree  of  temperature  to  which  milk  is  subjected?    dura- 
tion of  pasteurization  temperatures?    presence  or  absence 
of  air?    pressure,  whether  atmospheric  or  greater?    viscos- 
ity or  other  changes  in  milk  or  cream? 

What  changes  are  accomplished  by  pasteurization? 
Why  is  milk  pasteurized?  Is  this  end  always  accomplished 
in  commercial  plants? 

What  different  methods  are  used  commercially  for  the 
pasteurization  of  milk?  What  are  the  advantages  and 
disadvantages  of  each  method?  Why? 

At  what  stage  in  the  process  of  production  should 
milk  be  pasteurized  to  accomplish  the  desired  results? 
Must  the  after-treatment  of  pasteurized  milk  be  any  differ- 
ent from  that  of  unpasteurized  milk? 

Do  you  think  that  milk  should  be  pasteurized  before 
it  reaches  the  consumer? 

Does  pasteurization  affect  the  digestibility  of  milk? 
What  are  the  limitations  of  pasteurization  as  applied  to 
milk? 

17.  Give  your  results  in  full  and  any  conclusions  that 
may  be  drawn. 


284  GENERAL  MICROBIOLOGY 

REFERENCES 

MARSHALL:    Microbiology  (1911),  pp.  319-321. 

ROSENAU:    The  Milk  Question  (1912),  pp.  16,  37,  76,  105,  112,  120, 

128,  132,  138,  161,  185-230,  294. 
WARD:    Pure  Milk  and  the  Public  Health  (1909),  pp.  71,  73,  74,  114- 

125, 


EXERCISE  7.     DETERMINATION  OF  THE  NUMBER  AND 
TYPES  OF  BACTERIA  IN  BUTTER 

Apparatus.  Three  tubes  litmus  lactose  agar;  litmus 
milk  tubes;  fresh  butter;  sterile  dilution  flasks;  three 
sterile  Petri  dishes;  sterile  1  c.c.  volumetric  (bulb)  pipettes. 

Method.  1.  Melt  a  small  quantity  of  butter  in  a 
test  tube  at  the  lowest  possible  temperature  (not  higher  than 
40°  to  45°  C.).  Mix  well. 

2.  Using  a  warm  pipette,  transfer  1   c.c.  of  the  well- 
mixed  melted  butter  to  99  c.c.  of  sterile  (warm)  salt  solu- 
tion.    Free  the  pipette  from  fat  by  filling  it  with  the  dilu- 
tion water  several  times.      Use  warm   (50°   C.)    pipettes 
and  dilution  flasks  throughout  so  that  the  butter  will  not 
stick  to  the  pipettes  and  may  be  readily  emulsified. 

3.  Plate  in  litmus  lactose  agar,  using  dilutions  1  :  1,000, 
1  :  100,000  and  1  :  1,000,000. 

Note.  These  dilutions  may  have  to  be  changed.  Look  up  the 
average  number  of  bacteria  in  the  type  of  butter  you  are  using  and 
make  dilutions  accordingly. 

4.  Incubate  the  plates  at  25°  C. 

5.  Weigh  1  c.c.  of  well-mixed  melted  butter  and  record 
the  weight  in  grams. 

6.  Examine  the  plates  after  three  to  five  days  for  acid 
and  other  types  of  colonies. 

7.  Count  and  record  the  number  of  bacteria  per  cubic 
centimeter,  also  the  types.     Note  the  action  of  each  type 
on  litmus  milk. 

8.  Estimate  the  number  of  bacteria  per  gram. 


NUMBER  AND  TYPES  OF  BACTERIA  IN  BUTTER    285 

9.  What  is  the  melting-point  of  butter?    Are  bacteria 
ordinarily  killed  at  this  temperature? 

What  kinds  of  microorganisms  are  found  in  fresh  butter 
from  ripened  cream?  In  old  butter?  In  fresh  oleomar- 
garine? In  renovated  butter?  In  canned  butter? 

Do  bacteria  increase  or  decrease  in  butter  kept  in  stor- 
age? What  other  methods  of  making  a  bacteriological 
examination  of  butter  may  be  employed? 

Are  microorganisms  in  any  way  responsible  for  the 
flavors  of  butter?  Explain. 

What  pathogenic  organisms  may  gain  entrance  to 
butter? 

What  is  the  avenue  of  entrance?  How  long  can  bacteria 
exist  in  butter?  How  do  bacterial  numbers  and  types 
compare  with  those  of  fresh  milk?  of  ripened  cream? 

10.  Give  your  data  and  conclusions  in  full  and  point  out 
any  practical  applications, 

REFERENCES 

RUSSELL  and  HASTINGS:     Practical  Dairy  Bacteriology,  pp.  95,  97. 

LOHNIS:  Laboratory  Methods  in  Agricultural  Bacteriology,  pp. 
81-82. 

MARSHALL:     Microbiology,  pp.  335-345. 

SAYER,  RAHN  and  FARRAND:  Keeping  Qualities  of  Butter,  I.  Gen- 
eral Studies,  Tech.  Bui.  I  (1908),  Mich.  Agrl.  Expt.  Sta. 

RAHN,  BROWN  and  SMITH:  Keeping  Qualities  of  Butter,  II  and  III. 
Tech.  Bui.  2  (1909),  Mich.  Expt.  Sta. 

WELLS,  LEVI:  Renovated  Butter:  Its  Origin  and  History.  Year- 
book of  Dept.  of  Agr.  for  1905,  pp.  393-398. 

ROGERS,  L.  A.:  Studies  Upon  the  Keeping  Quality  of  Butter,  I. 
Canned  Butter.  Bui.  57,  B.  A.  I.,  U.  S.  Dept.  of  Agr.  (1904), 
pp.  6-8,  22-23. 


286  GENERAL  MICROBIOLOGY 


EXERCISE    8.     TO    DETERMINE    THE    NUMBER    AND 
TYPES  OF  MICROORGANISMS  IN  CHEESE 

Apparatus.  Cheddar  cheese;  cheese  trier  (sterile)  if 
an  uncut  cheese  is  to  be  sampled;  mortar,  with  pestle; 
two  knives,  sterile;  quartz  sand;  sterile  filter  papers  about 
6  and  8  cm.  square;  one  dilution  flask  containing  95  c.c. 
of  0.85%  salt  solution;  dilution  flasks,  containing  90  and 
99  c.c.  sterile  0.85%  salt  solution;  sterile  1  c.c.  and  10  c.c. 
pipettes;  three  tubes  litmus  lactose  agar;  three  sterile 
Petri  dishes;  sterile  litmus  milk  tubes. 

Method.  1.  Sterilize  in  the  hot-air  oven,  10  gms.  of 
sand  in  a  mortar  with  a  pestle. 

2.  Using  a  red-hot  knife  blade,  sear  a  portion  of  the  sur- 
face of  the  cheese. 

3.  To  weigh  the  cheese  "  aseptically,"  place  the  smaller 
sterile  filter  paper  upon  the  larger  on  the  balance  pan. 

h  4,  With  a  sterile  knife  remove  the  inner  portion  of  the 
seared  surface  and  obtain  and  weigh  out  5  gms.  of  cheese, 
using  aseptic  precautions. 

5.  Transfer  the  cheese  to  the  sterile  mortar  and  grind 
up  well. 

8.  Transfer  the  cheese  and  sand  mixture  with  sterile 
knife  or  spatula  to  the  95  c.c.  dilution  flask.  Shake  well  to 
free  the  sand  from  the  cheese. 

Directions  for  making  dilutions.  In  transferring  with 
a  pipette  a  portion  of  the  first  suspension  to  other  dilution 
flasks  the  sample  should  be  taken  immediately  after  shaking 
before  the  sand  has  settled.  Settling  may  be  avoided  by 
holding  the  pipette  in  a  horizontal  position  until  ready  to 
deliver  the  contents.  The  grinding  material  should  be  fine 
enough  to  avoid  clogging  the  pipette. 

7.  Make  and  plate  the  following  dilutions  in  litmus 
lactose  agar:  1  :  100,000;  1  :  10,000,000  and  1  :  1,000,000,- 
000,  if  the  cheese  has  been  made  recently. 

If  the  cheese  is  not  perfectly  fresh,  use  dilutions  1  :  100,- 


TYPES  OF  MICROORGANISMS  IN  CHEESE        287 

000;  1:1,000,000  and  1:10,000,000.  Lower  dilutions 
may  be  necessary  if  the  cheese  has  been  stored  for  some 
time. 

8.  Incubate  the  plates  at  25°  C.  for  three  to  five  days. 

9.  Count  and  estimate  the  number  of  microorganisms 
per  gram  of  cheese.     What  types  predominate?     Why? 

10.  Transfer  the  different  types  to  litmus  milk  and  note 
the  action  after  several  days.     Which  of  these  types,  as 
determined  from  the  action  on  litmus  milk,  may  have  a 
prominent  part  to  play  in  the  ripening  of  the  cheese?     Why? 

12.  Do  microorganisms  play  any  part  in  the  formation 
of  the  flavor  of  cheddar  cheese?     Other  cheeses? 

How  does  the  cheese  analyzed  compare  with  the  butter 
analyzed  as  to  numbers  and  types  of  microorganisms? 

What  qualitative  tests  are  made  for  milk  used  for  cheese 
making?  What  is  the  principle  and  application  of  these 
tests? 

What  cheese  "  abnormalities "  may  be  caused  by 
microorganisms?  During  what  stages'  in  the  process  of 
making  cheese  may  these  occur? 

What  pathogenic  organisms  have  been  found  in  cheese? 
What  is  known  of  their  longevity  in  this  medium? 

13.  State  your  results  and  conclusions  in  full  and  point 
out  any  practical  applications. 

REFERENCES 

LOHNIS:  Laboratory  Methods  in  Agricultural  Bacteriology,  pp.  83-88. 
RUSSELL  and  HASTINGS:  Experimental  Dairy  Bacteriology,  pp. 

103-109. 

MARSHALL:     Microbiology,  pp.  346-357. 
CONN:     Practical  Dairy  Bacteriology  (1907),  pp.  223-259. 
SAVAGE:     The  Bacteriological  Examination  of  Food  and  Water  (1914), 

pp.  118-119. 
DECKER:    Cheese  Making  (1905),  pp.  48-51,  63,  66,  69,  75,  88,  108- 

110. 
VANSLYKE  and  PUBLOW:    The  Science  and  Practice  of  Cheese-making 

(1912),  pp,  115-135,  285-312,  371-378, 


288  GENERAL  MICROBIOLOGY 


EXERCISE  9.  A  COMPARISON  OF  THE  BACTERIAL 
CONTENT  OF  SWEETENED  AND  UNSWEETENED 
CONDENSED  MILKS 

Apparatus.  Six  sterile  Petri  dishes;  six  tubes  of  litmus 
lactose  agar;  99  c.c.  dilution  flasks;  two  95  c.c.  dilution 
flasks;  tubes  of  sterile  litmus  milk;  two  sterile  5  c.c.  pipettes 
with  large  aperture  for  delivery;  can-opener. 

Culture.  Unopened  can  of  sweetened  condensed  milk; 
unopened  can  of  unsweetened  condensed  milk  (contents 
guaranteed  to  be  sterile) . 

Method.     1.  Sterilize  the  can-opener  in  the  flame. 

2.  Thoroughly  cleanse  the  outside  of  the  unopened  cans 
of  condensed  milk  and  then  submerge  in  boiling  water  for 
five  or  ten  minutes. 

3.  Remove  the  cans  from  the  water,  being  careful  in 
handling  them  not  to  contaminate  the  upper  surface  of  the 
cans. 

4.  With  the  sterile  can-opener  make  an  opening  in  the 
can  only  large  enough  to  admit  the  introduction  of  a  5  c.c. 
pipette. 

Note.  Only  one  can  is  to  be  opened  at  a  time  to  avoid  contam- 
ination. ' 

5.  With  a  sterile  pipette  obtain  a  5  c.c.  sample  from  the 
can  just  opened  and  transfer  to  a  95  c.c.  dilution  flask. 

Note.  As  the  condensed  milk  is  very  viscous  and  adheres  to  tho 
sides  of  the  pipette,  after  delivering  the  5  c.c.  into  the  dilution  flask 
blow  out  the  remainder  into  the  sink  or  other  suitable  place,  then 
replace  in  the  dilution  flask  and  wash  out  the  adhering  fluid  by  draw- 
ing the  diluting  fluid  up  into  the  pipette  several  times.  The  use  of  a 
5  c.c.  volumetric  pipette  having  a  large  aperture  for  delivery  would 
lessen  the  possibilities  of  contamination. 

6.  This  resulting  dilution  is  a  1  :  20  dilution  of  the  con- 
densed milk,  or  a  1  :  40  dilution  of  the  original  milk  (if 
the  directions  on  the  can  give  a  dilution  of  1  :  1  for  pro- 
ducing a  milk  of  original  composition). 


TYPES  OF  MICROORGANISMS  IN  ICE  CREAM  289 

7.  Plate  the  following  dilutions  of  the  condensed  milk 
in   litmus   lactose   agar:     1  :  20,    1  :  2000   and    1  :  20,000. 
Place  plates  at  25°  C. 

8.  Examine  and  count  at  the  end  of  five  days. 

9.  Record,  the  numbers  and  types  of  organisms  develop- 
ing on  the  plates.     Are  any  acid  colonies  present?     Deter- 
mine the  morphology  of  the  acid  colonies. 

10.  Transfer  each  type  of  colony  to  a  tube  of  sterile 
litmus  milk  and  observe  action  from  day  to  day.     Are  the 
types  which  are   present  desirable?     Is  Bad.   lactis  acidi 
present?     Any  organisms  of  the  B.  coli  type?     Are  patho- 
genic bacteria  apt  to  be  present? 

11.  To  what  factors  are  due  the  keeping  qualities  of 
each  type  of  condensed  milk? 

What  care  should  be  taken  of  opened  cans  of  milk  of 
either  type?  Of  the  milk  after  it  has  been  diluted  accord- 
ing to  directions? 

In  what  other  forms  is  concentrated  milk  sold?  What 
factors  are  responsible  for  the  keeping  quality  of  each  of 
these  latter  types? 

12.  Give  your  results  and  conclusions  in  detail, 

REFERENCES 

MARSHALL:     Microbiology  (1911),  363-366. 

SAVAGE:     The  Bacteriological  Examination  of  Food  and  Water  (1914), 

pp.  111-113. 
SADTLER:     Industrial  Organic  Chemistry  (1912),  pp.  281,  288. 

EXERCISE    10.     TO    DETERMINE    THE    NUMBER    AND 
TYPES  OF  MICROORGANISMS  IN  ICE  CREAM 

Apparatus.  Litmus  lactose  agar  shake;  three  tubes 
sterile  litmus  lactose  agar;  three  sterile  Petri  dishes;  ster- 
ile 1  c.c.  pipettes;  sterile  dilution  flasks;  sterile  wide- 
mouthed  glass-stoppered  bottle;  sterile  butter  trier;  sterile 
knife. 

Culture.     From  ice  cream. 


290  GENERAL  MICROBIOLOGY 

Method.     1.  Remove    the    (frozen)    ice    cream    sample 
from  the  container  by  means  of  the  sterile  butter  trier. 

2.  With  the  sterile  knife  discard  the  upper  portion  of  the 
sample  and  place  in  the  sterile  wide-mouthed  bottle. 

Note.     Pack  the  sample  in  ice  if  it  cannot  be  examined  at  once. 

3.  To  examine,  allow  the  ice  cream  to  melt  quickly  by 
placing   it   at    about    37°    C.    and   then   treat   as  a  milk 
sample. 

4.  Plate  on  litmus  lactose  agar,  using  the  following  dilu- 
tions:    1  :  10,000,    1  :  1,000,000    and    1  :  100,000,000    and 
incubate  plates  at  37°  C. 

5.  Add  a  large  quantity  (25  c.c.  to  50  c.c.)  to  the  melted 
agar  shake  and  incubate  at  37°  C.     Examine  in  twenty- 
four  to  forty-eight  hours   for   acid    and    gas.      Is  B.  coli 
present? 

6.  Count  plates  at  the  end  of  three  days  and  estimate 
the  total  number  of  bacteria  present  per  cubic  centimeter, 
also  the  number  of  acid  colonies  and  of  any  other  predom- 
inant type. 

7.  Transfer  predominant  types  to  litmus  milk  tubes  and 
note  action,  also  note  rapidity  with  which  each  type  pro- 
duces changes  in  the  litmus  milk.     What  may  these  results 
signify? 

8.  Make  a  microscopic  count,  using  the  method  in  Exer- 
cise 1,  Dairy  Microbiology.    How  do  microscopic  and  plate 
counts  compare? 

9.  Look  up  references  for  ascertaining  bacteriological 
standards    for   ice   creams.     What    is    the    quality  of  the 
ice  cream  you  analyzed  as  compared  with  the  maximum 
bacterial  limit?     What  do  you  think  this  limit  should  be? 

10.  From  what   diverse    sources  do  bacteria  enter  ice 
cream? 

What  is  their  significance  in  this  product? 
What  relation  may  some  of  the  common  practices  of 
ice-cream  makers  have  to  the  bacterial  content  of  milk? 


PLANTS   SUBJECT  TO  MICROBIAL  DISEASES      291 

What  effect  does  storage  have  upon  the  number  of  bac- 
teria in  properly  hardened  ice  cream? 

What  significance  has  a  pure  ice-cream  supply  in  relation 
to  public  health? 

11.  Give  results  and  conclusions  in  detail. 

REFERENCES 

SAVAGE:     The  Bacteriological  Examination  of  Food  and  Water  (1914), 

pp.  119-121. 

MARSHALL:     Microbiology  (1911),  pp.  372-373. 
WILEY,  H.  W.:     Ice  cream,  Hygienic  Lab.  Bui.  56.'     Milk  and  its 

Relation  to  the  Public  Health  (1909),  pp.  251-311. 
HAMMER,  B.  W.:     Bacteria  and  Ice  Cream,  Bui.  134  (1912),  Iowa 

Agr.  Expt.  Sta. 
MORTENSEN,   M.   and  GORDON,   J.:     Lacto:    a  New  and  Healthful 

Frozen    Dairy    Product.      Bui.    119    (1911),    Iowa   Agr.    Expt. 

Sta. 
WASHBURN,    R.    M.:  Principles   and  Practice  of  Ice-cream  Making. 

Bui.   155.    Vermont  Agr.  Expt.   Sta.,   pp.  9-10,   34-46,   53-54, 

64-66. 

BOLDUAN:    Food  Poisoning  (1909),  pp.  84-90. 
AYERS,  S.  H.  and  JOHNSON,  Jr.,  W.  T.:  A  Bacteriological  Study  of 

Retail  Ice  Cream,     Bui,  303  U.  S.  Dept.  Agr.,  1915. 

PLANT  MICROBIOLOGY 

EXERCISE  1.  TO  DEMONSTRATE  THAT  PLANTS  ARE 
SUBJECT  TO  MICROBIAL  DISEASES:  INFECTION 
OF  CERTAIN  SPECIES  OF  VEGETABLES  HAVING 
JUICY  ROOTS,  LEAVES,  FRUITS,  ETC.,  WITH  B. 
CAROTOVORUS 

Apparatus.  Tubes  of  sterile  2%  saccharose  broth; 
tubes  of  sterile  agar;  sterile  water;  sterile  Petri  dishes; 
three  sterile  deep  culture  dishes;  sterile  filter  paper;  sterile 
knife;  sterile  forceps;  mercuric  chloride,  1  :  500;  juicy 
vegetables. 

Culture.  B.  carotovorus  (culture  of  high  physiological 
efficiency). 

Method.  1.  The  root  of  the  carrot,  turnip,  rutabaga, 
the  cucumber  or  radish;  the  cotyledons  of  immature  pea 


292 


GENERAL  MICROBIOLOGY 


seedlings,  petioles  of  cabbage  seedlings,  potatoes,  etc., 
may  be  used  for  this  exercise.  For  what  other  plants  is 
B.  carotovorus  pathogenic? 

2,  Thoroughly  wash  the  root,   or  vegetable  to  be  in- 


FIG.  62. — Crown  Gall  Produced  by  Bact.  tumefaciens.     (Orig.) 

oculated.     Two  or  three  vegetables  of  one  kind  should  be 
employed. 

3.  Disinfect  a  spot  about  2  cm.  in  diameter  with  1  :  500 
mercuric  chloride  and  rinse  with  sterile  water  to  get  rid 
of  disinfectant.     Drain  off  excess  moisture  on  sterile  filter 
paper,  handling  vegetable  with  sterile  forceps. 

4.  Puncture  the  disinfected  area  on  one  vegetable  with 


PLANTS  SUBJECT  TO  MICROBIAL  DISEASES      293 

the  sterile  stiff  needle  for  control  and  place  in  a  sterile 
deep  culture  dish. 

5.  Obtaining  some  of  the  culture  of  B.  carotovorus  on 
the  sterile  needle,  puncture  the  remaining  vegetables  in  the 
center  of  the  disinfected  area  and  place  vegetables  in  a 
sterile  deep  culture  dish  at  20°  to  25°  C. 

B.  carotovorus  is  a  wound  parasite  which  invades  the 
intercellular  spaces,  dissolving  the  middle  lamellae  and 
portions  of  the  inner  lamellae,  thereby  establishing  a  con- 
dition which  is  known  as  soft  rot. 

6.  Examine  in  twenty-four  hours  for  evidence  of  action 
of  B.   carotovorus.     This  should   be   easily   distinguishable 
in  three  days. 

7.  Isolate  the  causal  organism  and  determine  its  mor- 
phology and  cultural  characteristics.     Compare  with   pure 
culture  and  with    description  given  in  Marshall's  Micro- 
biology, p.  512. 

Is  the  organism  newly  isolated,  capable  of  producing 
infection?  Make  inoculations  from  one,  two,  three  and 
four-day  old  newly  isolated  cultures  to  sterile  living  vege- 
table tissue  to  determine  this.  Is  there  any  difference  in 
the  infectivity  of  a  one-day  old  and  a  three-  or  four-day  old 
culture? 

8.  What  is  known  of  methods  of  control  of  this  disease? 

9.  Grow  four  giant  colonies  of  B.  carotovorus  on  ordinary 
agar,  one  in  each  Petri  dish  and  allow  them  to  develop 
until  nearly  1  cm.  in  diameter. 

10.  Under    sterile    conditions,    remove    slices    of    fresh 
carrot,  beet  and  rutabaga  or  turnip  roots  and  potato  and 
place  in  sterile  Petri  dishes.     (Slices  should  be   at  least 
3  to  4  cm.  in  diameter.) 

11.  With  a  sterile  scalpel  make  a  circular  incision  0.5 
cm.  from  the  edge  of  the  colony  through  the  layer  of  agar 
in  the  Petri  dish. 

12.  Remove  this  colony  intact  to  the  surface  of  one  of 
the  slices  of  vegetable  and  replace  cover  of  Petri  dish. 


294  GENERAL  MICROBIOLOGY 

» 

13.  Repeat,  removing  a  colony  to  the  slice  of  each  of  the 
different  vegetables. 

14.  Examine  in  twenty-four  hours  for  evidences  of  soft 
rot,  and  note  progress  of  softening  from  day  to  day.     What 
is  demonstrated  by  this  phenomenon?     Are  all  vegetables 
attacked? 

15.  What  parts  of  the  plant  does  B.  carotovorus  attack? 
What  chemical  constituents  of  these  parts  are  decomposed 
through  the  agency  of  their  action? 

What  are  the  main  features  of  difference  in  the  mechanism 
of  action  of  the  various  types  of  bacterial  plant  diseases? 

Give  an  example  of  a  disease  illustrating  each. 

How  is  the  progress  of  infection  effected  in  these  various 
types?  What  organisms  produce  a  disease  of  similar  type 
in  other  vegetables  and  plants? 

What  is  known  of  immunity  in  the  plant  kingdom? 

What  methods  of  control  are  employed  with  different 
types  of  plant  diseases?  How  are  methods  of  control 
influenced  by  the  type  of  disease? 

Note.  This  exercise  may  be  made  more  interesting  and  instructive 
if  combined  with  histological  methods. 

Plates  illustrating  the  invasion  of  root  tissues  by  B.  carotovorus 
are  found  in  Smith's  Bacteria  in  Relation  to  Plant  Diseases,  Vol.  I, 
pp.  56,  103. 

16.  State  in  full  your  results  and  conclusions. 

REFERENCES 

SMITH,  ERWIN  F.:  Bacteria  in  Relation  to  Plant  Diseases,  Vol.  I, 
pp.  5,  6,  65,  86,  103.  Vol.  II,  pp.  51-52,  65,  81-88,  96,  292. 
Vol.  III. 

MARSHALL:     Microbiology,  pp.  490-519. 

JONES,  L.  R.:  A  soft  rot  of  carrot  and  other  vegetables,  pp.  299, 
13th  Rept.  of  Vt.  Expt.  Sta.  (1901).  Also  in  Cent.  f.  Bakt.  II, 
Bd.  14,  pp.  369-377. 

JONES,  L.  R.:  Pectinase,  the  cytolytic  enzyme  produced  by  B.  caroto- 
vorus and  certain  other  soft-rot  organisms.  Tech.  Bui.  11,  N.  Y. 
Agr.  Expt.  Sta,  (1909), 


ANIMAL  INOCULATION  IN  BACTERIOLOGY       295 


ANIMAL   DISEASES    AND   IMMUNITY 

EXERCISE  1.  ANIMAL  INOCULATION  IN  BACTERIOL- 
OGY FOR  DETERMINATION  OF  THE  IDENTITY 
OF  A  MICROORGANISM,  ITS  PATHOGENICITY  OR 
VIRULENCE,  OR  FOR  PRODUCTION  OF  IMMUNITY 

Apparatus.  Experimental  animals:  rabbits;  guinea 
pigs;  white  rats;  white  mice,  etc.;  scalpels;  scissors; 
forceps;  razor;  syringe;  trephine;  sterile  dishes;  anes- 
thetic; disinfectant;  cotton. 

Culture.     Pure  culture  or  infected  material. 

I.     INTRODUCTION 

1.  Avoid  the  use  of  animals  where  the  employment  of 
other  means  answers  the  purpose  equally  well. 

2.  Unless  other  factors  prevent,  always  use  the  most 
susceptible  and  least  expensive  animals. 

II.     PREPARATION  OF  ANIMAL  * 

Method.  1.  (a)  Examine  carefully  each  animal  before 
subjecting  it  to  experimentation. 

(6)  Use  no  animal  already  showing  symptoms  of  illness 
or  general  lack  of  vigor. 

(c)  Record  the  weight  and  temperature  of  each  animal. 

2.  (a)  Administer  an  anesthetic  (general  or  local  as 
indicated)  whenever  the  operation  is  very  painful  or  tedious 
or  where  perfect  immobility  of  the  parts  is  required. 

Note.  For  local  anesthesia  a  2%  solution  of  cocaine  hydrochloride 
may  be  made  by  dissolving  0.1  gm.  of  cocaine  hydrochloride  in  5  c.c. 
of  sterile  water.  Instill  a  few  drops  into  the  conjunctiva!  sac  or  inject 
1  to  5  c.c.  into  the  subcutaneous  tissues  near  the  seat  of  operation. 
For  general  anesthesia  10  to  30  c.c.  of  a  5%  solution  of  chloral  hydrate 
may  be  injected  per  rectum,  or  ether  or  chloroform  may  be  inhaled. 

*The  instructor  must  arrange  for  experiments  that  must  be  started 
early  in  order  to  be  completed  before  the  term  closes. 


296 


GENERAL  MICROBIOLOGY 


Ether  is  probably  safer  in  the  hands  of  a  novice.  It  may  be  adminis- 
tered by  saturating  cotton  placed  in  a  paper  cone  which  is  kept  over 
the  animal's  nose.  Care  should  be  exercised  to  replenish  the  supply 
of  the  anesthetic  on  the  cotton  as  fast  as  it  volatilizes  and  not  to  force 
the  anesthetizing  too  fast.  Injury  to  the  integument  about  the 
nose  may  be  avoided  by  rubbing  on  vaseline  before  beginning  the 
operation.  The  tissues  should  not  be  cut  until  anesthesia  is  complete. 

(6)  Choose  a  site  for  operation  where  the  results  will  not 
interfere  with  the  animal's  locomotion  or  normal  functions, 
(c)  Use  sharp,  sterile  instruments. 


FIG.  63. — Tray  for  Sterilizing  Surgical  Instruments. 


Note.  Methods  for  holding  different  animals  for  different  forms 
of  operations  vary.  An  assistant  is  usually  required  to  hold  the  animal, 
where  an  anesthetic  is  not  administered,  and  where  an  anesthetic  is 
used  it  is  usually  better  to  have  an  assistant  administer  it,  although 
this  is  not  necessary.  (For  various  devices  for  holding  experimental 
animals  see  text-book:  Eyre,  Bacteriological  Technic,  2d  Ed.  (1913), 
pp.  349-352.) 

3.  Remove  the  hair  with  scissors  or  clippers  from  the 
field  of  operation  and  shave  the  surface.  Wash  the  skin 
and  disinfect  it  with  2%  liquor  cresolis  compositus  (U.  S.  P.). 
Wash  off  the  disinfectant  with  alcohol  and  allow  the 


ANIMAL  INOCULATION  IN  BACTERIOLOGY       297 

alcohol  to  evaporate.     The   animal  is  now  ready  for  the 
operation. 

Note.  It  is  understood  that  a  2%  solution  of  liquor  cresolis  com- 
positus  (U.  S.  P.)  shall  be  used  wherever  a  disinfectant  solution  is  indi- 
cated unless  otherwise  stated. 

III.     METHODS 

Where  the  exact  nature  of  the  inoculum  is  unknown, 
the  experimenter  will  be  guided,  as  to  what  method  to  select, 
by  his  judgment,  influenced  by  experience  with  other  inocula 
in  animal  experimentation.  The  method  most  adaptable 
in  the  case  of  each  specific  microorganism  will  be  indicated 
in  the  treatment  of  that  organism. 

1.  Cutaneous.    Rub  the  inoculum  on  the  shaved  and 
disinfected   skin  or   make  several  parallel,  superficial  inci- 
sions and  rub  the  inoculum  into  the  scarifications  with  a 
sterile  scalpel.     See  that  no  disinfectant  remains  on  the 
skin  before  operating. 

2.  Subcutaneous.     I.  (a)  Pick    up    the    skin    with    the 
thumb  and  forefinger  of  the  left  hand  and  insert  the  needle 
through  one  side  of  the  fold  of  skin  thus  made. 

Note.  The  point  of  the  needle  should  not  enter  the  skin  on  the 
other  side  of  the  fold,  but  should  lie  in  the  subcutaneous  tissue. 

(6)  Release  the  skin  and  inject  the  material. 

(c)  Place  the  finger  moistened  with  the  disinfectant 
over  the  point  where  the  needle  enters  the  skin  and  remove 
the  needle. 

II.  (a)  For  solid  material  that  will  not  pass  through  a 
hypodermic  needle,  make  a  short  incision  through  the  skin 
parallel  to  the  horizontal  plane  of  the  body. 

(b)  With  a  sterile  probe  separate  the  skin  from  the 
underlying   tissues   on   the    lower    side    of   the   cutaneous 
incision,    making    a    small    pocket   in    the    subcutaneous 
tissue. 

(c)  With  fine-pointed  sterile  forceps  insert  the  inoculum 


298  GENERAL  MICROBIOLOGY 

into  this  pocket.     Further  treatment  should  not  be  neces- 
sary. 

3.  Intramuscular.     I.  Plunge  the  needle  deeply  into  the 
muscles,  preferably  on  the  inside  of  the  thigh. 

II.  Inject  the  material  slowly  with  steady  pressure  if 
the  volume  is  great. 

4.  Intravenous.    I.  Inject  the  liquid  into  the   ear  vein 


FIG.  64. — One  Method  of  Injecting  Hog-cholera  Serum.     (Orig.) 

of  the  rabbit  (and  other  animals  if  possible)  or  jugular  vein 
where  accessible.  The  injection  should  be  in  the  direction 
of  the  circulation. 

II.  The  femoral  vein  may  be  used  where  other  veins 
are  not  readily  entered  with  the  needle.  Use  general 
anesthesia.  Make  an  incision  on  the  inside  of  the  thigh 
over  the  femoral  space.  Separate  the  iliacus,  pectineus 
and  sartorius  muscles.  The  femoral  vein  and  artery  are 
laid  bare.  After  inoculation  disinfect  and  suture  the  skin. 


ANIMAL  INOCULATION  IN  BACTERIOLOGY       299 

Note.  Solid  substances,  larger  than  leucocytes,  and  air  bubbles 
should  not  be  injected  into  the  vascular  system.  Fatal  emboli  may 
result.  Return  of  blood  through  the  needle  indicates  that  the  vein 
has  been  entered.  If  swelling  occurs  at  point  of  inoculation  the  inocu- 
lum is  entering  the  subcutaneous  tissue.  Try  again. 

6.  Intraabdominal  or  intraperitoneal.  I.  The  site  for 
the  operation  is  the  center  of  the  angle  formed  by  the  last 
rib,  transverse  processes  of  the  lumbar  vertebrae,  and  the 
external  angle  of  the  ilium. 

(a)  Plunge  the  needle  or  trocar  and  canula  through  the 
abdominal  wall  with  one  thrust. 

Note.  When  the  parietal  peritoneum  is  punctured  the  sudden 
disappearance  of  resistance  to  the  entrance  of  the  needle  is  noticed. 
The  intestines  will  not  be  entered  if  pressure  on  the  needle  stops  at 
this  point. 

(6)  Inject  the  material  and  remove  the  needle,  placing 
the  thumb  and  finger  on  each  side  of  the  needle  and  press- 
ing gently  on  the  skin  during  the  removal  so  as  to  prevent 
separating  the  skin  and  underlying  layers  of  tissue. 

II.  Infectious  material  or  cultures  in  a  sterile  collodium 
capsule  may  be  introduced  into  the  abdominal  cavity  by 
performing  laparotomy.  (General  anesthesia  is  desired.) 

6.  Intraorbital.     Always  perform  under  local  anesthesia. 
(2%  cocaine  hydrochloride.) 

I.  Steady  the  eye  with  fixation  forceps. 

II.  Pierce  the  cornea  near  to  its  periphery  with  a  fine 
needle.     The  needle  should  incline  with  the  point  outward 
so  that,  upon  entering  the  anterior  chamber  of  the  eye, 
the  iris  will  not  be  damaged. 

III.  Inject  the  material. 

7.  Subdural.     Operate  under  general  anesthesia. 

I.  Make  a  longitudinal  incision  through  the  skin  at  one 
side  of  the  sagittal  suture.     Hold  back  the  skin  and  sub- 
cutaneous tissue  with  tenacula. 

II.  Make  a  crucial  incision  through  the  periosteum  and 
push  back  the  four  corners. 


300 


GENERAL  MICROBIOLOGY 


III.  Expose  the  dura  mater  by  removing  a  small  button 
of  the  parietal  bone  (0.5  cm.  in  diameter)  with  a  trephine. 

IV.  Inject  the  inoculum  immediately  beneath  the  dura 
mater. 

V.  Replace  the  periosteum  and  suture  the  skin. 

VI.  Disinfect. 

8.  Intrapulmonary.     I.  Pull  the  animal's  front  leg  for- 
ward. 


FIG.  65. — Another  Method  of  Injecting  Hog- cholera  Serum,     (Orig.) 

II.  Plunge  the  needle  through  the  fifth  or  sixth  inter- 
costal space  into  the  lung  tissue. 

III.  Slowly  inject  the  contents  of  the  syringe. 

Note.  In  large  animals  material  may  be  injected  into  the  trachea 
between  the  tracheal  rings. 

9.  Ingestion.  If  possible  mix  the  infectious  material 
with  the  animal's  food. 

Note.     See  that  the  animal  eats  all  that  is  intended  to  be  eaten. 

Introduce  the  infectious  material  into  a  gelatin  capsule 
and  force  the  animal  to  swallow  it;  or  give  the  material  as 
a  drench  where  advisable. 


ISOLATION  OF  PATHOGENIC  BACTERIA          301 

Note.  Fasting  the  animal  before  introducing  unpalatable  material 
into  the  food  may  be  helpful  in  increasing  the  amount  eaten.  The 
chemical  reaction  of  the  stomach  contents  as  governed  by  physio- 
logical activity  will  influence  results. 

IV.  CARE  OF  INOCULATED  ANIMALS 

1.  Watch  each  animal  closely  and  take  temperatures  as 
the  case  demands. 

2.  Treat  each  animal  as  a  case  of  infectious  disease  in 
quarantine. 

Note.  Whatever  clinical,  diagnostic  or  sanitary  measures  neces- 
sary in  that  given  disease  may  be  employed  as  seen  fit. 

3.  When  the  animal  is  removed  from  the  cage  for  the 
last  time,  carefully  destroy  all  refuse  in  the  cage  and  dis- 
infect thoroughly. 

4.  Give   all   results,   observations    and    conclusions    in 
detail. 

REFERENCES 

STITT:    Practical   Bacteriology,    Blood    Work   and   Parasitology,   3d 

Ed.,  1914,  pp.  48,  143,  152. 
MOORE  and  FITCH:     Bacteriology  and  Diagnosis,  pp.  114,'  118-120, 

124,  125,  130,  133. 

EYRE:     Bacteriological  Technic.  pp.  332-369. 
KOLMER:  Infection,  Immunity  and  Specific  Therapy  (1915),  pp.  53-64. 

EXERCISE  2.  THE  ISOLATION  OF  PATHOGENIC  BAC- 
TERIA FROM  FLUIDS  AND  TISSUES  OF  DEAD 
ANIMALS 

Apparatus.  Disinfectant;  scalpel;  scissors;  forceps; 
bone  forceps;  ten  sterile  pipettes;  10  c.c.  sterile  pipettes; 
250  c.c.  flask  containing  glass  beads,  sterile;  sterile  Esmarch 
dishes;  spatula;  platinum  loop;  special  media. 

Method.     1.  Disinfect  the  skin. 

2.  Remove  the  spleen,  kidney,  lymph  glands,  and  any 
other  diseased  tissue,  to  sterile  Esmarch  dishes,  using  sterile 
instruments. 


302  GENERAL  MICROBIOLOGY 

3.  Collect   samples    of    pericardial    and  pleuritic  fluids* 
blood,    urine    and    bile    with    sterile    pipettes    and    place 
these  in  small  sterile    flasks.     Collect    at  least   25    c.c.    of 
blood  in  a  sterile  flask  containing  glass  beads  for  defibrinat- 
ing. 

4.  Remove  the  organs  collected  to  the  laboratory  and 
make  cultures  as  follows: 

5.  Sear  the  surface  of  the  organ  with  a  spatula  heated  to 
a  white  heat. 

6.  Tear  the  seared  surface  with  forceps,  sterilized  in 
flame. 

7.  With  a  sterile  platinum  loop,  make  transfers  to  agar 
slants,  shake  cultures,  and  plates  for  isolation   into    pure 
cultures. 

8.  Repeat  7,  using  any  body  fluids  collected. 

Note.  The  different  diseases  require  special  procedures  and 
media  for  successful  results.  Attention  will  be  called  to  these  varia- 
tions at  the  proper  places. 

REFERENCES 

MOORE  and  FITCH:     Bacteriology  and  Diagnosis,  pp.  95-96. 
MOORE:    Principles  of  Microbiology,  pp.  156-162,  237-258. 
EYRE:     Bacteriological  Technic,  pp.  248-258. 


EXERCISE  3.     A  STUDY  OF  BACT.  ANTHRACIS 

Note.  Bact.  anthracis  is  the  cause  of  anthrax,  a  disease  very  fatal 
to  man  and  certain  domestic  animals.  Great  care  should  be  taken 
while  working  with  it. 

Apparatus.  Six  tubes  of  agar;  three  tubes  of  potato; 
three  tubes  of  milk;  tube  of  gelatin;  slides  and  stains; 
autopsy  instruments. 

Culture.     Bact.  anthracis. 

Method.  1.  Inoculate  three  tubes  each  of  agar,  potato^ 
and  milk  and  one  tube  of  gelatin  with  Bact.  anthracis. 


THE  PREPARATION  OF  TUBERCULIN     303 

2.  Incubate  one  tube  of  each  at  20°  C.,  one  at  37°  C., 
and  one  at  42°  C.     (Study  and  record  the  effect  of  these 
temperatures  upon  the  growth  and  spore  formation  of  the 
organism.) 

3.  Make  cover-glass  preparations  and  stain  with  meth- 
ylen    blue,   fuchsin  and  Gram's   stain.      Stain    for  spores 
(Anjeszky's  method). 

4.  Transfer  a  small   quantity  of  the   agar   culture   to 
4   or    5    c.c.    of    sterile    physiological    salt    solution    and 
inject  0.25  c.c.  subcutaneously  into  a  guinea  pig.     Make 
daily    observations    and     an    autopsy   of    the    animal   at 
death. 

5.  Make  cultures  on  agar  slants,  and  smear  prepara- 
tions from  the  blood,  liver,  spleen  and  kidney  after  the 
autopsy. 

6.  Fix  the  smears  in  the  flame,  stain  with  methylen  blue 
or  fuchsin.      After  twenty-four  and    forty-eight  hours  ex- 
amine the  cultures  microscopically. 

7.  State  your  results  and  conclusions  in  full. 

REFERENCES 

MARSHALL:  Microbiology,  pp.  469,  476,  559,  561,  599-604. 
JORDAN:     General  Bacteriology,  4th  Ed.  (1914),  pp.  223-236. 
BESSON:     Practical  Bacteriology,  Microbiology  and  Serum  Therapy, 

transl.  by  Hutchens  (1913),  pp.  517-535. 
KOLMER:   Infection,  Immunity  and  Specific  Therapy  (1915),  pp.  653- 

654. 
ZINSSER:  Infection  and  Resistance  (1914),  pp.  15,  18,  53,  64,  296. 

EXERCISE  4.  THE  PREPARATION  OF  TUBERCULIN 

Apparatus.  Two  500  c.c.  Erlenmeyer  flasks;  glycerin- 
ated  veal  broth;  evaporating  dish;  0.5%  phenol  salt  solu- 
tion; Berkefeld  filter;  heavy  filter  paper;  20  c.c.  homeo- 
pathic vials;  sealing  wax. 

Culture.  Bact.  tuberculosis  (specially  adapted  for 
tuberculin) . 

Method,     1.  Place  about  200  c.c.  of  glycerinated  veal 


304 


GENEKAL  MICROBIOLOGY 


bouillon  in  each   of  two  500  c.c.    Erlenmeyer  flasks  and 
sterilize  for  twenty  minutes  each   day   for  three  consecu- 
tive days. 

R2.  From  a  culture  of  the  tuber- 
cle bacterium  furnished,  inoculate 
each  flask  of  veal  broth.  In  mak- 
ing the  inoculations  care  should 
be  taken  to  place  the  inoculum  on 
the  surface  and  to  avoid  agitation 
after  inoculation.  Seal  the  flasks 
with  paraffin  and  place  in  the  incu- 
bator at  a  temperature  of  37°  C. 

3.  Allow  the   cultures   to   grow 
four    weeks    after    the    surface    is 
covered,  then  shake  well,  place  in 
a  steam  sterilizer  and  subject  to 
steam  for  two  and  a  half  hours. 

4.  Filter  through  a  filter  paper 
to   remove   most    of   the  bacterial 
growth. 

5.  Evaporate    to    one-tenth  its 
original  volume  over  a  water  bath 
at  a  temperature  of  60°  C. 

6.  To  one  volume  of   the  con- 
centrated   tuberculin     add     seven 
volumes    of    sterile     physiological 
salt     solution      containing     0.5% 
phenol  or  tricresol  and  then  filter 
through  a  Berkefeld  filter. 

7.  Place  the  product  in  20  c.c. 
homeopathic  vials  and  seal  with  wax.     Label  the  vials  and 
place  in  a  cool  dark  room. 


FIG.  66. — Bact.  tuberculosis 
(avian),  on  Banana. 
(Orig.  Himmelberger.) 


THE  PREPARATION  OF  TUBERCULIN 


305 


REFERENCES 

MARSHALL:  Microbiology,  pp.  485,  487. 

MOORE:     Principles  of  Microbiology,  pp.  251-253. 


FIG.  67.— Glycerin  Veal-broth  Cultures  of  Bad.  tuberculosis  (Human), 
for  Tuberculin,  about  Eight  Weeks  Old,     (Orig.  Keck,) 

BESSON:     Practical  Bacteriology,  Microbiology  and/ Serum  Therapy, 

pp.  289-345. 

MOORE:  Bovine  Tuberculosis. 
KOLMER:  Infection,  Immunity  and. Specific  Therapy  (1915),  pp.  582- 

601,  661-680. 
ZINSSER:  Infection  and  Resistance  (1914)  ,pp.  355-357,  438-442. 


306 


GENERAL  MICROBIOLOGY 


THE  PREPARATION  OF  BLACK-LEG  VACCINE     307 


EXERCISE  5.     THE  PREPARATION  OF  BLACK-LEG  VAC- 
CINE 

Apparatus.  Sterile  mortar  and  pestle;  sterile  cheese- 
cloth; two  sterile  glass  plates;  sterile  water;  sterile  homeo- 
pathic vials. 

Culture.  Diseased  muscle  of  calf  affected  with  black- 
leg. 

Method.  1.  Place  a  piece  of  diseased  muscle  from  a 
calf  affected  with  black  leg  (this  will  be  furnished  by  the 
instructor)  in  a  sterile  mortar,  add  a  small  quantity  of 
water  and  triturate  completely  with  a  sterile  pestle. 

2.  Squeeze  the  pulp  through  a  piece  of  sterile  cheese 
cloth,  spread  the  filtrate  in  a  thin  layer  over  a  sterile  glass 
plate  or  saucer  and  dry  at  a  temperature  of  35°  to  37°  C. 
in  an  atmosphere  free  from  contamination. 

3.  Mix  approximately  one  part  by  volume  of  the  dried 
virus  with  two  parts  of  water,  triturate  until  the  mixture 
is  converted  into  a  semi-solid  homogeneous  mass  and  spread 
in  a  thin  layer  over  a  glass  plate  or  saucer. 

4.  Heat  in  an  oven  to  a  temperature  of  100°  to  104°  C. 
for  a  period  of  seven  hours. 

5.  One  centigram  of  the  attenuated  virus  mixed  with  a 
small  quantity  of  water  is  a  dose  for  a  calf.     Place  the 
product  in  sterile  vials,  ten  doses  to  the  vial,  place  a  cork 
stopper  in  each  vial  and  seal. 

REFERENCES 

NORGAARD,  V.  A.,  and  MOHLER,  J.  R.:     Black  leg,  its  Nature,  Cause 

and  Prevention,  B.  A.  I.  Circular  No.  31,  revised  (1911). 
MARSHALL:     Microbiology,  pp.  472-473. 
KOLMER:  Infection,  Immunity  and  Specific  Therapy  (1915)  p.  655. 


308  GENERAL  MICROBIOLOGY 


EXERCISE  6.     THE  PREPARATION  OF  TETANUS  TOXIN 

Apparatus.  Dextrose  broth;  two  100  c.c.  Erlenmeyer 
flasks;  paraffin  oil;  5%  phenol;  Berkefeld  filter. 

Culture.     B.  tetani. 

Method.  1.  Place  50  c.c.  of  dextrose  bouillon  in  each 
of  two  100  c.c.  Erlenmeyer  flasks,  plug  and  boil  gently 
two  or  three  minutes  over  a  flame. 

2.  Cover  the  bouillon  with  a  layer  of  paraffin  oil  about 
5  mm.  deep  and  heat  in  the  autoclav. 

3.  After  cooling,  inoculate  the  bouillon  with  B.  tetani 
and  incubate  about  two  weeks. 

4.  Examine   the   culture   microscopically   to   determine 
the  absence  of  contamination,  add    sufficient  5%  phenol 
to  make  a  0.5%  solution  and  filter  through  a  Berkefeld  filter. 

5.  Incubate  about  1  c.c.  of  the  filtrate  in  10  c.c.  of 
dextrose  broth  under  anaerobic  condition,  for  forty-eight 
hours  to  make  sure  that  the  filtrate  is  sterile. 

6.  The  filtrate,  if  sterile,  is  to  be  used  in  immunizing  a 
rabbit  for  the  production  of  antitoxin. 

Note.  A  fairly  potent  toxin  will  kill  a  guinea  pig  in  a  0.001  c.c. 
dose.  The  toxin  in  solution  is  very  unstable  and  should  be  kept  in  a 
tightly  stoppered  bottle  in  a  cool  dark  place.  It  may  be  kept  for 
several  months  by  precipitating  with  a  saturated  solution  of  ammo- 
nium sulphate  and  drying  in  vacuo  over  sulphuric  acid. 

REFERENCES 

MARSHALL:     Microbiology  (1911),  pp.  480-484. 

BESSON:     Practical  Bacteriology,  Microbiology  and  Serum  Therapy 

(1913),  pp.  536-548. 
KOLMER:    Infection,  Immunity  and  Specific  Therapy  (1915),  pp.  115, 

234,  720,  819. 
ZINSSER:  Infection  and  Resistance  (1914),  pp.  41,  107,  131-133. 


PREPARATION  OF  TETANUS  ANTITOXIN         309 


EXERCISE  7.     THE  PREPARATION  OF  TETANUS  ANTI- 
TOXIN 

Note.  In  the  preparation  of  tetanus  antitoxin  for  therapeutic 
purposes,  healthy  horses  are  used.  For  the  first  injection  of  toxin 
a  small  fraction  of  a  cubic  centimeter  is  given  subcutaneously.  The 
increase  in  the  size  of  the  dose  and  the  frequency  of  injection  depend 
upon  the  condition  of  the  animal,  but  the  quantity  injected  is  gradually 
increased  until  the  animal  is  able  to  stand  300  to  400  c.c.  of  toxin  at 
one  injection. 

For  laboratory  purposes,  the  rabbit  may  be  used  to  furnish  the 
antitoxin. 

The  following  method  suggested  by  Roux  and  Vaillard 
produces  a  satisfactory  antitoxin  for  laboratory  study. 

Apparatus.  Tetanus  toxin;  Gram's  iodin  solution; 
rabbits;  20  c.c.  syringe;  disinfectant;  anesthetic;  oper- 
ating tray;  50  c.c.  sterile  glass  cylinder. 

Method.  1.  Give  the  first  five  or  six  injections  sub- 
cutaneously, subsequent  ones  may  be  given  intraperi- 
toneally. 

1st  day,  3  c.c.  of  toxin  mixed  with  1  c.c.  of  Gram's  iodin 
solution. 

5th  day,  5  c.c.  of  toxin  mixed  with  2  c.c.  of  Gram's  iodin 
solution. 

9th  day,  12  c.c.  of  toxin  mixed  with  3  c.c.  of  Gram's 
iodin  solution. 

16th  day,  5  c.c.  of  undiluted  toxin. 

23d  day,  10  c.c.  of  undiluted  toxin. 

30th  day,  15  c.c.  of  undiluted  toxin. 

The  quantity  may  be  gradually  increased  until  the 
rabbit  is  getting  100  c.c.  of  undiluted  toxin. 

2.  After  the  6th  injection  has  been  given,  wait  a  period 
of  ten  days  and  bleed  the  rabbit  aseptically.  This  is  accom- 
plished as  follows: 

(a)  Secure  the  rabbit  in  a  dorsal  position  on  an  operating 
tray  and  anesthetize  with  ether. 

(b)  Expose  an  area  about  3  cm.  square  over  the  inferior 


310  GENERAL  MICROBIOLOGY 

thoracic  wall,  in  the  region  of  the  apex  of  the  heart,  shave 
and  clean  with  alcohol. 

(c)  Insert  a  sterile  needle  attached  to  a  sterile  20  c.c. 
syringe,  through  the  thoracic  wall  into  the  heart  and  slowly 
draw  the  plunger  out. 

If  only  a  small  quantity  of  serum  is  desired  for  testing, 
the  animal  may  be  saved  for  subsequent  bleedings. 

(d)  Place   the   blood   in   a   sterile   container,    allow   to 
clot  and  draw  off  the  serum  for  standardization. 

REFERENCES 

MARSHALL:    Microbiology  (1911),  pp.  480-484. 

BESSON:     Practical  Bacteriology,  Microbiology  and  Serum  Therapy 

(1913),  pp.  544-548. 
KOLMER:    Infection,  Immunity  and  Specific  Therapy  (1915),  pp.  234, 

242,  719-729. 
ZINSSER:  Infection  and  Resistance  (1914),  p.  463. 

EXERCISE    8.     A   DEMONSTRATION    OF   THE   AGGLU- 
TINATION TEST 

Note.  There  are  two  methods  of  applying  the  agglutination 
test:  First,  by  combining  the  suspect's  serum  in  varying  amounts 
with  a  suspension  of  the  specific  organism  and  incubating  eighteen  to 
thirty-six  hours;  the  results  are  then  read  with  the  unaided  eye. 
Second,  the  serum  may  be  combined  in  varying  dilutions  with  a  sus- 
pension of  the  specific  organism,  and  hanging  drop  preparations  made 
and  examined  microscopically.  If  agglutinins  are  present,  clumping 
of  the  organisms  will  occur  in  a  few  minutes.  With  either  method, 
controls,  containing  the  organism  but  normal  serum,  should  be  pre- 
pared for  comparative  purposes. 

Apparatus.  Four  agar  slants;  test-tube  rack  for  small 
test  tubes;  twelve  small  test  tubes;  antiserum;  physio- 
logical salt  solution;  1  c.c.  pipettes,  graduated  to  0.01  c.c.; 
5  c.c.  pipettes;  cover-glasses;  concave  slide. 

Culture.     B.  typhosus  or  B.  cholerce  suis. 

Method.  Macroscopic  Test.  1.  Antigen.  This  is  a 
suspension  of  the  specific  organism  obtained  from  a  twenty- 
four  to  forty-eight  hour  agar  culture  in  physiological  salt 


DEMONSTRATION  OF  AGGLUTINATION  TEST       311 

solution.  Only  a  sufficient  quantity  of  the  growth  to  give 
a  slight  cloudiness  to  the  salt  solution  in  a  small  test  tube 
should  be  used. 

Note  on  Antigen.  Where  a  series  of  agglutination  tests  are  to 
be  made  at  intervals,  the  antigen  should  be  standardized  so  that 
the  same  concentration  will  be  used  for  each  test.  Great  care  should 
be  used  in  preparing  the  antigen  to  avoid  clumps  in  suspension. 
In  some  cases  thoroughly  shaking  in  a  shaking  machine  will  afford  a 
satisfactory  antigen,  in  others  it  must  be  filtered  through  a  filter  paper, 


FIG.  69. — Macroscopic  Agglutination  of  B.  cholerce  suis  by  Dorset- 
McBryde-Niles  Serum.  From  left  to  right  tubes  show,  first, 
complete  agglutination,  heavy  sediment,  clear  supernatant 
liquid;  in  each  succeeding  tube  the  sediment  becomes  less,  the 
turbidity  greater,  the  tube  at  the  right  showing  uniform  cloudi- 
ness and  no  sediment,  no  agglutination.  (Orig.  Giltner.) 

2.  The   antiserum   may    consist   of   immune   serum — a 
rabbit  immunized  to  the  typhoid  bacillus  may  be  used  to 
furnish  the  serum — ,  or  hog  cholera  serum  or  virus  may  be 
used  with  B.  typhosus  and  B.  cholerce  suis  respectively. 

3.  The  following  table  shows  the  various  combinations 
of  serum,  antigen  and  salt  solution  to  give  definite  dilu- 
tions.    Physiological  salt  solution  should  be  used  in  dilut- 
ing the  serum. 


312 


GENERAL  MICROBIOLOGY 


Tube. 

Antigen. 

Serum 
Diluted  1-10. 

Salt  Solution. 

Dilution. 

c.c. 

c.c. 

c.c. 

1 

4 

1.0 

0.0 

1-50 

2 

4 

0.5 

0.5 

1-100 

3 

4 

0.2 

0.8 

1-250 

4 

4 

0.1 

0.9 

1-500 

Serum 

diluted  1-100. 

5 

4 

0.5 

0.5 

1-1000 

6 

4 

0.2 

0.8 

1-2500 

7 

4 

0.1 

0.9 

1-5000 

4.  Shake  all  tubes  well  and  incubate  at  37°  C.  for  twenty- 
four  hours  and  record  the  results. 

Microscopic  Test.  1.  If  this  test  is  carried  out  during 
the  same  period  with  the  macroscopic  test,  a  small  loopful 
of  the  dilution  from  any  tube  may  be  transferred  to  a  clean 
cover-glass  placed  on  a  hanging  drop  slide  and  the  edges 
sealed  with  vaselin  or  oil.  It  may  then  be  examined  with 
a  microscope. 

2.  If  done  independently  of  the  macroscopic  test,  prepare 
the  suspension  of  organisms  in  one  test  tube  and  the  dilu- 
tions of  serum  in  others. 

Mix  a  loopful  of  the  diluted  serum  with  a  loopful  of  the 
antigen  on  a  clean  cover-glass,  mount  on  a  concave  slide 
and  observe  with  a  microscope  for  a  period  of  thirty  minutes 
to  one  hour. 

3.  Give  results  and  any  conclusions  in  detail. 

REFERENCES 

KOLMER:  Infection,  Immunity  and  Specific  Therapy  (1915),  pp.  68, 

69,  79,  152,  266-291. 

MARSHALL:     Microbiology  (1911),  pp.  488,  567-570. 
MCFARLAND:     Pathogenic  Bacteria  and  Protozoa,   7th  Ed.    (1912), 

pp.  149-152. 
GELTNER:    Studies  of  Agglutination  Reactions  in  Hog  Cholera  during 

the   Process   of   Serum   Production    (Preliminary)    Tech.  Bui.  3 

(1909),  Mich.  Agr.  Expt.  Sta. 

GILTNER:     Same  title  Tech.  Bui.  8  (1911),  Mich.  Agr.  Expt.  Sta. 
ZINSSER:  Infection  and  Resistance  (1914),  pp.  218-247. 


A  STUDY  OF  FILTERABLE  VIRUSES  313 


EXERCISE  9.     A  STUDY  OF  FILTERABLE  VIRUSES 

Apparatus.  Physiological  salt  solution;  Chamberland 
filter  with  water-suction  or  air  pump  and  pressure  gage; 
sterile  flasks;  clinical  thermometer;  syringe;  flasks  of  bouil- 
lon, 50  c.c.  in  each;  autopsy  set. 

Culture.  Hog  cholera  virus  (blood  of  hog  sick  with 
cholera) . 

Method.  1.  Preparation  of  the  Filter.  If  the  filter  has 
been  used  once  clean  it  by: 

(a)  First  rinsing  with  cold  water  under  the  tap. 

(6)  Force  about  1  liter  of  cold  distilled  water  through 
it. 

(c)  Then  a  solution  consisting  of  1  gm.  KMn(>4  and  6.5 
gms.  HC1  in  1000  gms.  water. 

(d)  Next,  1000  c.c.  of  a  solution  of  1%  oxalic  acid. 

(e)  Boiling  water  is  then  forced  through  the  filter  until 
the  liquid  which  runs  through  is  free  from  acid. 

(/)  Lastly,  cold  distilled  water  must  be  forced  through 
the  filter. 

Thus  treated,  any  organic  residue  is  destroyed  and  the 
filter  is  as  good  as  new. 

This  method  of  purification  must  always  be  used  imme- 
diately after  using  a  filter.  Filter  candles  must  not  be  left 
twenty-four  hours  without  cleaning. 

A  new  filter  may  be  prepared  for  use  by  forcing  through 
it  a  large  quantity  of  boiling  distilled  water  and  finally 
cold  distilled  water. 

The  amount  of  liquid  necessary  to  force  through  the 
filter  for  cleaning  varies  with  the  size  of  the  filter.  The 
ordinary  8  inch  filter  should  receive  the  full  amount 
(1000  c.c.)  of  each  solution  and  distilled  water  for  efficient 
purification. 

Filters  are  best  sterilized  by  being  set  up  ready  to  use 
and  autoclaved.  (See  Fig.  71  for  one  method.) 

2.  Procure  some  hog  cholera  virus  and  after  diluting  it 


314 


GENERAL  MICROBIOLOGY 


t "  : ; , .•,^nir] 


s 

I 
I 

•s 

'I 
fr 


A  STUDY  OF  FILTERABLE  VIRUSES 


315 


with   equal   parts   of   physiological   salt   solution,    pass   it 
through  a  clean,  sterile,  Chamberland  filter  at  a  pressure 
not  to  exceed  one   atmosphere  and 
during  a  time    not    to   exceed   one 
hour. 

3.  Make  sub-cultures  of  the  fil- 
trate by  introducing  1  c.c.  into  each 
of  several  flasks  of  bouillon  contain- 
ing 50  c.c.  each.     Take  every  pre- 
caution against  contamination.  Also 
make  microscopical  preparations. 

4.  If  no   growth  results  under  2 
inject   2  c.c.  into   the   muscles  of  a 
50  Ib.  pig.     Make  daily  observations 
of  the  pig  and  record  the  temperature 
each  day, 

6.  When  undoubted  symptoms 
of  hog  cholera  have  developed,  kill 
the  pig  and  make  a  careful  autopsy. 
Save  the  blood  in  a  sterile  jar. 

6.  Repeat  the  experiment,  using 
blood  procured  in  4  as  virus. 

7.  By  repeated  nitrations  and  in- 
jecting   into    susceptible    hogs,    it 
may  be  proven  that  a  living  micro- 
organism, incapable  of  producing   visible  growth  in  vitro, 
passes  through  the  filter  and  develops  in  the  body  of  the 

pig- 

8.  State  your  results  and  conclusions  in  full, 


FIG.  71. — Pasteur-Cham- 
berland  Filter  Adjusted 
for  Filtration  by  Suc- 
tion. 


REFERENCES 

DORSET,  MCBRYDE  and  NILES:  Further  Experiments  Concerning 
the  Production  of  Immunity  from  Hog  Cholera,  Bui.  102,  B.  A.  I., 
U.  S.  Dept.  Agr. 

MCBRYDE:  Filtration  Experiments  with  B.  choleras  swis,  Bui.  113, 
B.  A.  I.,  U.  S.  Dept.  Agr. 


316  GENERAL  MICROBIOLOGY 

GILTNER:  What  is  the  Antigen  Responsible  for  the  Production  of 
Antibodies  in  Hog  Cholera  Serum?  Tech.  Bui.  13,  Mich.  Agr. 
Expt.  Sta. 

KOLMER:  Infection,  Immunity  and  Specific  Therapy  (1915),  pp.  77,  78. 


EXERCISE  10.     THE  PREPARATION  OF  BACTERINS  OR 
BACTERIAL  VACCINES 

Apparatus.  Scalpel;  scissors;  forceps;  sterile  tubes 
and  Esmarch  dishes;  sterile  swabs;  sterile  physiological 
salt  solution;  50%  alcohol;  disinfectant;  four  agar  slants; 
six  agar  tubes  for  plating;  six  sterile  Petri  dishes. 

Culture.  Infected  material  or  specific  cultures  to  be 
furnished  by  the  instructor. 

A.     AUTOGENOUS  BACTERINS 

Method.  In  the  preparation  of  an  autogenous  bac- 
terin,  it  is  first  necessary  to  isolate  the  microorganism 
causing  the  disease.  This  is  accomplished  as  follows: 

1.  If  there  are  any  unopened  abscesses,  open  one  with 
a  sterile  scalpel  after  first  disinfecting  the  field  with  2% 
compound  solution  of  cresol  an4  washing  with  50%  alcohol. 
Collect  some  of  the  pus  on  a  sterile  swab  and  suspend 
in  sterile 'physiological  salt  solution. 

2.  If   the   abscess   is   already   opened,    using   a   sterile 
curette,  obtain  some  of  the  diseased  tissue  at  the  bottom 
of  the  abscess  and  macerate  this  in  sterile  physiological 
salt  solution. 

3.  Pour  agar  plates  from  this  salt  solution  suspension, 
using  at  least  six,  plated  in  series  of  two. 

4.  Incubate  the    plates    and    after    twenty-four   hours 
make  observations  on  the  number  and  type  of  colonies. 
After  forty-eight  hours  make  transfers  to  agar  slants  of  the 
most  numerous  type  of  colony.     Colonies  should  be  studied 
under  low  power  of  the  microscope. 

5.  Grow  three  or  four  cultures  of  the  organism  on  slanted 


BACTERINS  OR  BACTERIAL  VACCINES  317 

agar.  Make  a  morphological  study  of  the  organism.  After 
twenty-four  hours  wash  off  the  growth  from  each  tube 
with  3  c.c.  of  sterile  saline  solution. 

6.  Put  the  suspension  all  in  one  container,  reserving  1 
c.c.  to  be  used  in  standardization. 

Note.  In  the  hemocytometer  method  for  standardizing  bacterins 
it  is  desirable  to  use  a  special  hemocytometer  with  a  counting  chamber 
0.02  mm.  deep  provided  with  a  special  cover-glass  for  counting  bac- 
teria, but  if  this  is  not  accessible,  an  ordinary  hemocytometer  and 
cover-glass  as  used  for  blood  counting  may  be  used.  If  the  latter, 
a  4  mm.  objective  must  be  used  for  counting. 

Using  the  diluting  pipette  of  the  blood  counting  apparatus  the 
suspension  of  bacteria  is  diluted  to  the  desired  dilution  with  Collison's 
fluid  made  as  follows: 

Hydrochloric  acid,  2  cc. 

Mercuric  chloride  1-500,  100  cc. 

•   Acid  fuchsin,  1%  aqueous  solution — enough  to  color  to  a  deep 
cherry  red. 

Filter  before  using. 

The  bacterial  suspension  is  allowed  to  remain  in  the  pipette  eight 
to  ten  minutes  to  stain,  then  thoroughly  agitated  by  rotating  the 
pipette  and  the  first  few  drops  from  the  arm  of  the  pipette  discarded. 
The  mount  is  then  prepared  and  the  slide  placed  on  the  stage  of 
microscope  which  has  been  previously  leveled,  and  the  count  made. 
The  count  and  calculations  are  made  as  for  blood  counting. 

7.  Heat  in  a  water  bath  at  60°  C.  for  one  hour.     This 
is  usually  sufficient  to  kill  the  bacteria,  unless  they  are 
spore  producers. 

8.  To  test  the  sterility  of  the  suspension  after  heating, 
with  a  sterile  loop  make  an  agar  streak  and  incubate  for 
twenty-four    hours.     If    growth    is    obtained    the    culture 
must  be  heated  again. 

B.     STOCK  BACTERINS 

The  procedure  in  the  preparation  of  a  stock  bacterin 
is  the  same  as  in  the  preparation  of  an  autogenous  bacterin, 
except  that  the  organisms  used  are  from  cultures  kept  in 
stock  for  that  purpose. 


318 


GENERAL  MICROBIOLOGY 


C.     POLYVALENT  BACTERINS 

Polyvalent  bacterins  are  those  which  are  prepared  from 
several  species  of  bacteria,  e.g.,  M.  (Staph.)  albus,  M. 
(Staph.)  aureus,  Strep,  pyogenes,  etc. 

The  suspension  of  each  must  be  prepared  and  standard- 


FIG.  72. — Blood-counting  Apparatus  for  Use  in  Standardizing  Bacterial 

Vaccines. 


ized  separately,  and  then  the  emulsions  of  all  mixed.  In 
this  way,  it  is  possible  to  have  a  known  number  of  each 
species  in  the  resulting  product. 

REFERENCES 

FITCH  C.  P.:  A  Review  of  the  Principal  Methods  Used  to  Standardize 

Bacterins  (Bacterial  Vaccines).     Report  of  the  N.  Y.  State  Vet. 

College  for  the  year  1913-1914,  pp.  207-219. 
MCCAMPBELL:     Laboratory  Methods  for  the  Experimental  Study  of 

Immunity,  pp.  186,  188. 
STITT:     Practical    Bacteriology,    Blood   Work    and   Parasitology,    2d 

Ed.  (1911),  pp.  143-145. 
KOLMER:  Infection,  Immunity  and  Specific  Therapy  (1915),  pp.  611- 

661. 
ZINSSER:  Infection  and  Resistance  (1914),  pp,  328-357. 


TO  DEMONSTRATE  OPSONINS  319 


EXERCISE  11.     TO  DEMONSTRATE  OPSONINS  AND  TO 
DETERMINE  THE  OPSONIC  INDEX 

Apparatus.  Several  .small  test  tubes;  forty-five  small 
mixing  pipettes;  sterile  citrated  salt  solution;  Wright's 
stain;  suspension  of  leucocytes;  normal  serum;  patient's 
serum. 

Culture.     Organism  producing  the  disease. 

Method.  1.  The  small  mixing  pipettes  are  made  by 
drawing  out  4  to  5  mm.  glass  tubing  to  a  long,  fine  capil- 
lary tube;  and  providing  with  a  small  rubber  bulb.  (Con- 
sult the  instructor  for  the  method.) 

2.  Prepare  the  suspension   of  leucocytes  by  collecting 
a  few  cubic  centimeters  of  blood  from  any  animal  and 
immediately  place  in  three  or  four  volumes  of  citrated  salt 
solution. 

Centrifuge  and  wash  at  least  three  times,  being  careful 
not  to  pipette  off  any  of  the  cells  during  the  washing 
process. 

After  the  last  washing,  pipette  off  the  supernatant 
liquid  and  lay  the  tube  in  as  nearly  a  horizontal  position 
as  possible  for  about  twenty-five  to  thirty  minutes.  At 
this  time  there  will  appear  an  upper  whitish  layer  of  cells 
composed  almost  exclusively  of  leucocytes. 

Pipette  off  the  leucocytes.  They  should  be  used  within 
five  to  six  hours  from  the  time  the  blood  is  collected. 

3.  Bacterial  Suspension.     Transfer   a  loopful  from   an 
eighteen  to  twenty-four  hour  agar  culture  to  2  or  3  c.c.  of 
physiological  salt  solution  and  mix  well.     The  suspension 
must  be  carefully  made  to  avoid  clumps  and  some  method 
of  standardization  used  so  that  successive   tests  will  be 
comparable.     (The    nephelometer    may    be    used    for    this 
purpose — see  an  instructor.) 

4.  Collect  the  patient's  serum  and  normal  serum  at  the 
same  time  and  under  the  same  conditions  in  order  that  the 
results  may  be  comparable.     The  blood  is  collected  in  a 


320  GENERAL  MICROBIOLOGY 

small  test  tube  and  either  allowed  to  clot  and  the  serum 
removed,  or  it  is  defibrinated  and  centrifuged.  In  either 
case  the  serum  should  be  used  within  three  to  four  hours 
from  the  time  the  blood  is  drawn. 

5.  Make  the  test  as  follows : 

a.  With  a  diamond  point  or  wax  pencil  make  a  mark  on 
the  drawn  out  arm  of  the  mixing  pipette  about  2  cm.  from 
the  end. 

b.  With  the  aid  of  a  rubber  bulb  on  the  opposite  end, 
draw  a  column  of  the  bacterial  suspension  up  to  the  mark, 
admit  a  bubble  of  air,  then  draw  a  column  of  leucocytes 
up  to  the  mark  and  another  bubble  of  air,  then  a  column 
of  the  serum  to  be  tested. 

c.  Mix  these  by  forcing  out  on  a  glass  slide  or  into  a 
small  test  tube  and  drawing  up  again,  repeating  once  or 
twice,  being  careful  to  avoid  introducing  air  bubbles. 

d.  Finally  draw  up  the  mixture  into  the  pipette  and  seal 
the  end  of  pipette  in  the  flame,  using  care  not  to  heat  the 
mixture. 

e.  Incubate  fifteen  minutes  with  frequent  shaking. 

/.  Then  place  a  drop  on  a  slide  and  make  a  thin  film 
made  as  in  the  preparation  of  a  blood  film,  dry  and  stain 
with  Wright's  or  Jenner's  stain. 

6.  Repeat  the  experiment,  using  the  normal  serum. 

7.  With  an  oil  immersion  lens   count  the  number  of 
organisms  taken  up  by  fifty  leucocytes  on  each  slide  and 
calculate   the   average   number   taken   up   by   each.     The 
result  is  the  opsonic  power  of  the  serum. 

8.  The  opsonic  index  is  the  ratio  of  the  opsonic  power 
of  the  suspected  serum  to  that  of  the  normal  serum. 

Example.  If  the  average  number  of  bacteria  taken  up 
by  the  leucocytes  in  the  presence  of  the  suspect  serum  is 
5.6  and  the  average  number  taken  up  by  the  leucocytes  in 
the  presence  of  the  normal  serum  is  4.8,  the  opsonic  index 
of  the  suspect  serum  is  determined  as  follows:  5.6^-4.8  = 
1.16+  —opsonic  index. 


TO  DEMONSTRATE  THE  PRECIPITIN  TEST       321 

McCampbell's  Modification  of  the   Opsonic  Test.     1. 

Prepare  the  bacterial  suspension  as  above  and  add  0.8% 
sodium  citrate. 

2.  (a)  With  a  blood  diluting  pipette,  draw  the  bacterial 
suspension  up  to  the  mark  0.5. 

(6)  With  the  same  pipette  draw  up  the  same  amount 
of  blood  collected  from  the  patient,  then  draw  both  into  the 
bulb  and  mix  quickly. 

(c)  Place  a  flat  rubber  band  around  the  ends  of  the 
pipette  and  incubate  fifteen  minutes.  Prepare  film,  and 
stain. 

3.  Repeat  the  experiment,   using  normal  blood.     The 
opsonic  index  is  determined  as  above. 

Note.  The  sodium  citrate  is  slightly  antiopsonic  but  this  factor 
is  constant  in  both  preparations,  consequently  the  results  are  com- 
parable. 

4.  Give  results  and  any  conclusions  in  detail. 

REFERENCES 

McCAMPBELL:     Laboratory    Methods    for    the   Experimental    Study 

of  Immunity  (1909),  pp.  44-70. 

MCFARLAND:     Pathogenic  Bacteria  and  Protozoa,  7th  Ed.,  p.  307. 
KOLMER:  Infection,  Immunity  and  Specific  Therapy,  pp.  187-205. 
ZINSSER    I.e.  Exercise  10,  p.  318. 

EXERCISE    12.     TO    DEMONSTRATE   THE   PRECIPITIN 

TEST 

This  test  is  of  importance  in  identifying  the  source  of 
blood  in  legal  cases  and  may  also  be  used  in  the  examina- 
tion of  various  meat  products  for  the  presence  of  foreign 
meat  substances. 

It  is  based  upon  the  fact  that  if  an  animal  is  injected 
at  intervals  of  six  to  eight  days  for  four  or  five  times  with 
any  foreign  protein  its  serum  acquires  the  property  of  pre- 
cipitating that  specific  protein  even  when  in  a  very  high 
dilution. 


322  GENERAL  MICROBIOLOGY 

An  antiserum  for  each  specific  protein  to  be  tested  for 
must  be  prepared  by  animal  inoculation.  Thus,  if  a  test 
for  human  blood  is  to  be  made,  an  anti-human-blood  serum 
must  be  used. 

Apparatus.  Syringe  and  needles;  sterile  cow's  blood; 
sterile  500  c.c.  Erlenmeyer  flask  containing  glass  beads; 
rabbit;  disinfectant;  sterile  flasks,  tubes  and  pipettes; 
sterile  physiological  salt  solution. 

Method.  1.  Inject  a  rabbit  intra-abdominally  with 
6  c.c.  sterile,  defibrinated  cow  blood.  On  the  sixth  or  seventh 
day,  repeat  the  injection,  using  10  c.c.  On  the  twelfth  or 
fourteenth  day  give  12  c.c.  and  again  on  the  eighteenth  or 
twenty-first  day  give  another  12  c.c. 

2.  On  the  twenty-fourth   or   twenty-eighth   day   draw 
a  little  blood  from  the  rabbit  and  test  it  to  determine  its 
property  of  precipitating  cow  blood.     If  it  has  a  high  litre, 
the  rabbit  should  be  anesthetized  and  the  bloo.d  drawn  from 
the  heart  as  explained  in  Exercises  1  and  7,  pp.  295  and  309. 

3.  Place  the  blood  in  a  sterile  container  and  allow  to 
clot.     Draw  the  serum  into  small,  sterile,  glass  bulbs  hold- 
ing 0.5  c.c.  and  seal  the  bulbs  by  heating  the  arm  in  a  small 
flame,  using  care  to  avoid  heating  the  serum.     Serum  col- 
lected in  this  way  and  placed  in  a  cool,  dark  place  will  retain 
its  precipitating  properties  for  several  months. 

4.  Dilute  the  blood  to  be  tested  with  physiological  salt- 
solution.     Several  dilutions  should  be  made,  e.g.,   1-200, 
1-500,  1-1000,  1-10,000  and  2  c.c.  of  each  dilution  placed 
in  small  test  tubes.     Place  six  to  eight  drops  of  the  anti- 
serum  in  each  tube.     If  the  suspect  serum  is  from  the  same 
species  of  animal  as  that  used  in  immunizing  the  rabbit 
(in   this   case,    cow   serum),   immediate   precipitation   will 
occur.     After  a  few  minutes'  observation  the  tubes  should 
be  incubated  at  37°  C.  for  twenty  to  thirty  minutes  and  the 
results  noted. 

5.  If  the  blood  is  dried,  as  a  blood  stain  on  cloth,  the 
quantity   should   be   estimated   and   placed   in   a   definite 


THE  PRODUCTION  OF  A  HEMOLYTIC  SERUM     323 

quantity  of  salt  solution  so  that  the  dilution  may  be  approx- 
imated. 

6.  Give  results  and  conclusions  in  full. 

REFERENCES 

MCFARLAND:  Pathogenic  Bacteria  and  Protozoa,  7th  Ed.,  p.  146. 

MARSHALL:     Microbiology  (1911),  pp.  570-574. 

NUTTALL:     Blood  Immunity  and  Relationship  (1904). 

KOLMER:  Infection,  Immunity  and  Specific  Therepy  (1915),  pp.  70,  71, 

292-315,  517-519,  844-846. 
ZINSSER:  Infection  and  Resistance  (1914),  pp.  248-271, 

EXERCISE    13.     THE  PRODUCTION  OF   A  HEMOLYTIC 

SERUM 

For  this  work  a  rabbit  will  be  immunized  to  washed 
sheep  blood  cells. 

Apparatus.  Sterile  physiological  salt  solution;  glass 
beads;  small  glass  funnel;  two  200  c.c.  Erlenmeyer  flasks; 
five  or  six  sterile  centrifuge  tubes;  sterile  5  c.c.  pipette, 
with  rubber  bulb  attached  for  draining  off  serum  and  salt 
solution  in  centrifuge  tubes;  sterile  14-gage  2j-inch  hypo- 
dermic needle ;  sheep;  rabbit. 

Note.  Chemically  pure  salt  and  distilled  water  should  be  used  in 
the  preparation  of  salt  solution  for  this  work  and  for  the  complement 
fixation  test. 

Method.  1.  In  one  Erlenmeyer  flask  place  eight  or 
ten  glass  beads  for  defibrinating  blood,  plug  and  sterilize 
in  hot  air. 

Provide  the  other  one  with  a  small,  sterile,  glass  funnel 
and  two  layers  of  sterile  cheese-cloth  for  filtering  and  de- 
fibrinating blood. 

2.  With  an  attendant  holding  the  sheep,  clip  the  wool 
over  the  area  of  the  jugular  vein  and  wash  with  50%  alcohol. 

3.  Place  the  thumb  over  the  jugular  furrow  about  half 
way  between  the  head  and  shoulder  and  press  on  the  vein 
so  that  it  will  become  distended. 


324  GENERAL  MICROBIOLOGY 

4.  Thrust  the  needle  through  the  skin  anterior  to  the 
thumb,  into  the  vein  and  draw  about  20  to  30  c.c.  of  blood 
into  the  flask  containing  the  glass  beads. 

5.  Defibrinate  by  agitating  three  or  four  minutes  and 
filter  through  two  layers  of  cheese-cloth. 

6.  Mix  the  blood  with  approximately  an  equal  amount 
of  sterile  physiological  salt  solution  and  centrifuge  in  sterile 
centrifuge  tubes. 

7.  Draw  off  the  supernatant  fluid  down  to  the  corpuscles, 
using  the  sterile  pipette  with  bulb  attached. 

8.  Fill    the   tube   with   sterile    salt    solution    and    mix 
thoroughly  by  pouring  from  one  tube  to  another  several 
times. 

9.  Centrifuge  again  and  repeat  6  and  7,  at  least  five  times. 

10.  Mix  the  washed  corpuscles  with  an  equal  volume  of 
sterile  physiological  s,alt  solution,  warm  to  body  tempera- 
ture and  inject  14  c.c.  into  the  peritoneal  cavity  of  a  rabbit. 

11.  Six  or  seven  days  later  inject  the  rabbit  intra-peri- 
toneally  with  20  c.c.  of  a  50%  suspension  of  washed  sheep 
blood  cells  and  again  on  the  fourteenth  day  with  24  c.c.  of 
a  50%  suspension. 

12.  About  a  week  or  ten  days  from  the  last  injection 
draw  a 'small  quantity  of  blood  from  the  rabbit,  allow  to 
clot,  pipette  off  the  serum  and  inactivate  at  a  temperature 
of  56°  C.,  for  thirty  minutes  and  titrate.     (For  method  of 
titration,  see  Exercise  No.  14.) 

Note.  Care  must  be  exercised  in  the  above  operations  to  avoid 
contaminating  the  blood  cells  and  they  must  be  thoroughly  washed 
and  injected  on  the  same  day  they  are  drawn. 

13.  State  in  full  your  results  and  any  conclusions  that 
may  be  drawn. 

REFERENCES 
See  Exercise  14. 


THE  COMPLEMENT  FIXATION  TEST  325 

EXERCISE  14.     TO  DEMONSTRATE  THE  COMPLEMENT 
FIXATION  TEST 

The  complement  fixation  test  is  one  of  the  most  com- 
plicated biological  reactions  used  as  a  means  of  diagnosis 
in  infectious  diseases. 

Apparatus.  Guinea  pig;  rabbit;  sheep;  suspected 
serum  (from  aborting  cow  or  other  animal  to  be  tested); 
small  test  tubes;  test-tube  rack;  flasks;  physiological 
salt  solution;  centrifuge  tubes;  disinfectant;  syringe  and 
needles. 

Culture.  Bad.  abortus  (or  other  organism  depending 
on  disease  for  which  test  is  made). 

I.    TITRATION  OF  REAGENTS 

Method.  Four  reagents  other  than  the  serum  to  be 
tested  are  required:  1,  complement;  2,  hemolysin;  3,  red 
blood  cells  from  a  sheep;  4,  antigen.  Above  components  1, 
2  and  4  must  be  titrated  before  using  in  order  to  deter- 
mine the  amounts  to  be  used  in  the  tests. 

1.  Complement.     This  is  contained  in  and  obtained  from 
fresh  serum  from  a  guinea  pig.     The  complement  is  titrated 
for  the  purpose  of  determining  the  least  amount  which  in 
the  presence  of  a  sufficient  amount  of  hemolysin  will  produce 
complete  hemolysis  of  a  definite  quantity  of  washed  red 
blood  cells  from  the  sheep.     This  amount  is  spoken  of  as 
the  litre. 

2.  Hemolysin  (see  Exercise  13).     The  source  of  hemo- 
lysin is  inactivated  serum  from   a  rabbit   that   has  been 
previously  immunized    to  washed  red  blood    cells  from  a 
sheep. 

The  selection  of  a  rabbit  and  sheep  is  merely  a  matter 
of  convenience.  Any  two  animals  of  a  different  genus 
may  be  used.  In  the  test  for  syphilis  in  man,  human 
blood  cells  are  usually  used  because  more  convenient  to 
obtain  in  a  number  of  laboratories. 


326 


GENERAL   MICROBIOLOGY 


TITRATION  OF  COMPLEMENT 


Tubes. 

i 

2 

3 

4 

5 

6 

7 

Salt  solution  

(A) 

c.c. 

1.5 

c.c. 

1.5 

c.c. 

1.5 

c.c. 

1.5 

c.c. 

1  5 

c.c. 
1  5 

c.c. 

1  5 

Hemolysin  
Suspension    of   blood 
corpuscles 

(£) 
(C) 

0.1 
0  5 

0.1 
0  5 

0.1 
0  5 

0.1 
0  5 

0.1 
0  5 

0.1 
0  5 

0.0 
0  5 

Complement  

(D) 

0.02 

0.04 

0.06 

0.08 

0.1 

0.0 

0.1 

Result  after  one-half 
hour  

(K) 

- 

* 

+ 

+ 

_!_ 

- 

-  _ 

Incubate  all  tubes  in  water  bath  at  37°  C.  for  one-half  hour  and  read. 

A.  0.9%  salt  solution. 

B.  1%  dilution  of  inactivated  immune  rabbit  serum  in  salt  solu- 
tion. 

C.  1%  suspension  of  washed  sheep-blood  cells  in  salt  solution. 

D.  20%  solution  of  fresh  guinea  pig  serum  in  salt  solution  (0.4 
c.c.  complement  made  up  to  2  c.c.  with  salt  solution). 

E.  *  A  variation  of  reaction  according  to  strength  of  complement, 
-f  =  complete  hemolysis. 

—  =no  hemolysis. 

The  inactivation  is  accomplished  by  heating  to  a  tem- 
perature of  56°  C.  for  one-half  hour  to  destroy  the  com- 
plement. If  not  used  for  several  days  it  is  not  necessary 
to  heat,  >  as  complement  is  destroyed  on  standing.  If  it 
is  to  be  kept  for  some  time,  preserve  by  adding  5% 
phenol  sufficient  to  make  a  0.5%  solution.  It  is  then 
titrated  to  determine  the  smallest  quantity  which  will  bring 
about  a  complete  solution  of  the  same  quantity  of  washed 
sheep  blood  cells  used  in  the  titration  of  the  complement, 
when  in  the  presence  of  a  proper  quantity  of  complement. 

3.  Antigen.  Antigen  is  an  extract  of  the  specific  bacteria 
made  by  growing  the  bacteria  on  agar  and  washing  off  with  a 
few  cubic  centimeters  of  salt  solution,  and  is  preserved  with 
phenol  sufficient  to  make  0.5%  and  glycerin  sufficient  to 
make  1%.  The  suspension  is  placed  in  a  shaking  machine 
for  three  hours  a  day  for  three  consecutive  days  to  obtain 
homogeneity. 


THE   COMPLEMENT  FIXATION   TEST 
TITRATION  OF  HEMOLYSIN 


327 


Tubes. 

l 

2 

3 

4 

5 

6 

7 

8 

c.c. 

c.c. 

c.c. 

c.c. 

c.c. 

c.c. 

c.c. 

c.c. 

Salt  solution  .  .  . 

(A] 

1.5 

1.5 

1.5 

1.5 

1.5 

1.5 

1.5 

1.5 

Hemolysin  

(B) 

0.01 

0.02 

0.04 

0.06 

0.1 

0.15 

0.15 

0.0 

Suspension  blood 

cells  

(O 

0.5 

0.5 

0.5 

0.5 

0.5 

0.5 

0.5 

0.5 

Complement.  .  .  . 

(D) 

0.1 

0.1 

0.1 

0.1 

0.1 

0.1 

0.0 

0.1 

Result  after  one- 

half  hour  

(E) 

* 

* 

* 

+ 

+ 

+ 

— 

— 

Incubate  all  tubes  in  water  bath  at  37°  C.  for  one-half  hour  and 
read. 

A.  0.9%  salt  solution. 

B.  1%  dilution  of  inactivated  immune  rabbit  serum  in  salt  solution. 

C.  1%  suspension  of  washed  sheep-blood  cells. 

D.  Titrated  guinea  pig  serum  diluted  so  that    0.1   c.c.  contains 
1.5  times  the  titre. 

E.  +  indicates  complete  hemolysis.    • 
—  indicates  no  hemolysis. 

*  indicates  a  variation  in  the  reaction  according  to  the  strength  of 
the  hemolysin. 

The  smallest  quantity  causing  complete  hemolysis  is  called  the 
titre. 

A  titration  of  this  reagent  is  made  to  determine  the 
smallest  quantity  that  will  prevent  hemolysis  in  the  presence 
of  1.5  times  the  titre  of  complement  and  the  hemolysin, 
sheep  cells  and  immune  serjim.  In  other  words,  we  must 
determine  the  smallest  quantity  of  antigen  that  will  fix 
the  amount  of  complement  used  in  the  test. 

TIRATION  OF  THE  ANTIGEN 


Tubes. 

1 

2 

3 

4 

5 

6 

7 

8 

c.c. 
1.5 
0.00 
0.15 
0.1 

9 

10 

11 

Salt  solution  
Positive  serum  .  .  . 

(A) 
(B) 
(C) 
(D) 

c.c. 
1.5 
0.02 
0.01 
0.1 

c.c. 
1.5 
0.02 
0.02 
0.1 

c.c. 
1.5 
0.02 
0.05 
0.1 

c.c. 
1.5 
0.02 
0.1 
0.1 

c.c. 
1.5 
0.02 
0.15 
0.1 

c.c. 

1.5 
0.02 
0.2 
0.1 

c.c. 
1.5 
0.02 
0.25 
0.1 

c.c. 
1.5 
0.00 
0.2 
0.1 

c.c. 
1.5 
0.00 
0.25 
0.1 

c.c. 
1.5 
0.00 
0.3 
0.1 

Complement  

Incubate  for  half  an  hour  in  a  water  bath  at  37°  C.,  then  add  the  hemolytic  sys- 
tem as  follows: 


328 


GENERAL  MICROBIOLOGY 


HEMOLYTIC  SYSTEM 


Tubes. 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

Hemolysin 

(E) 

c.c. 
0  1 

c.c. 
0  1 

c.c. 
0  1 

c.c. 
0  1 

c.c. 
0  1 

c.c. 
0  1 

c.c. 
0  1 

c.c. 
0  1 

c.c. 
0  1 

c.c. 
0  1 

c.c. 
0  1 

Suspension  of  blood 
cells 

(F) 

0  5 

0  5 

0  5 

0  5 

0  5 

0  5 

0  5 

0  5 

0  5 

0  5 

0  5 

Results     after     in- 

cubation   

(G) 

* 

* 

— 

— 

— 

— 

— 

+ 

+ 

+ 

+ 

Incubate  half  an  hour  in  a  water  bath  at  37°  C.  and  then  keep  in  the  ice  box  for 
twelve  hours  and  read. 

A.  0.9%  salt  solution. 

B.  Inactivated  serum  known  to  contain  antibodies. 

C.  Suspension  of  a  culture  of  suspected  bacteria,  carbolized. 

D.  Titrated  guinea  pig  serum  diluted  so  that  0.1  c.c.  contains  1.5  times  the 
titre. 

E.  Immune  rabbit  serum  of  known  titre  diluted  so  that  0.1  c.c.  contains  three 
times  the  titre. 

F.  1%  suspension  of  washed  sheep  blood  cells  in  salt  solution. 

G.  *  signifies  a  variable  reaction  according  to  the  activity  of  t 
+  signifies  a  complete  hemolysis. 

—  signifies  no  hemolysis. 

The  smallest  quantity  of  antigen  (in  combination  with  antibody)  that  com- 
pletely fixes  the  complement  is  known  as  the  litre. 


ity  of  the  antigen. 


II.    COMPLEMENT  FIXATION  TEST.     (Test  proper) 

1.  Suspected  Serum.   'This  is  drawn  from'  the  animal 
that   is   suspected   of  being  infected   with   the  infectious 
disease  in  question. 

The  blood  is  drawn  from  the  jugular  vein.  It  is  allowed 
to  clot  and  the  serum  collected.  It  must  be  inactivated 
before  testing  unless  it  is  to  be  held  for  a  week  or  more 
before  applying  the  test,  in  which  case  inactivation  is  not 
necessary,  but  1%  phenol  should  be  added  as  a  preserva- 
tive. 

Note.  The  test  proper  and  controls  must  be  run  at  the  same  time. 
If  several  tests  are  run  at  the  same  time  one  set  of  controls  is  sufficient. 

2.  Test  of  Suspect  Serum. 


Tubes. 

Test  Proper. 

Controls. 

1 

2 

3 

4 

1 

2 

3 

4 

Salt  solution  
Suspect  serum.  .  . 

(A) 

(B) 
(C) 
(D) 

::;:: 

c.c. 
1.5 
0.1 
0.1 
0.1 

c.c. 
1.5 
0.02 
0.1 
0.1 

c.c. 
1.5 
0.04 
0.1 
0.1 

c.c. 
1.5 
0.04 
0.1 
0.0 

c.c. 
1.5 
0.0 
0.0 
0.0 

c.c. 
1.5 
0.0 
0.0 
0.1 

c.c. 
1.5 
0.0 
0.1 
0.1 

c.c 
1.5 
0.0 
0.1 
0.0 

Complement  

Incubate  one-half  hour  at  37°  C,  in  water  bath. 


THE  COMPLEMENT  FIXATION  TEST 


329 


3.  Then  add  the  hemolytic  system. 


Tubes. 

Test  Proper. 

Controls. 

1 

2 

3 

4 

1 

2 

3 

4 

Blood  cells  
Hemolysin       .  .  . 

(E) 

(F) 

c.c. 
0.5 
0.1 

c.c. 

c.c. 

c.c. 

c.c. 

c  c 

c  c 

c.c. 
0.5 
0.1 

0.5 
0.1 

0.5 
0.1 

0.5 
0.1 

0.5 
0.0 

0.5 
0.1 

0.5 
0.1 

Incubate  one-half  hour  at  37°  C.  in  water  bath  and  then  place  in  ice  box  for 
twelve  hours  and  read. 

A.  Salt  solution,  0.9%., 

B.  Suspect  ser\im  inactivated  for  one-half  hour. 

C.  Antigen,  two  times  titre. 

D.  Complement,  1.5  times  titre. 

E.  1%  washed  sheep  blood  corpuscles  in  salt  solution. 

P.  Immune  rabbit  serum  (hemolysin)  diluted  so  that  0.1  c.c.   contains  three 
times  the  titre. 

Control  tubes  2  and  3  should  show  complete  hemolysis. 
Control  tubes  1  and  4  should  show  complete  absence  of  hemo- 
lysis. Control  tube  4  is  control  on  the  inactivation  of  the 
suspect's  serum  and  should  show  absence  of  hemolysis. 

Hemolysis  in  the  other  tubes  indicates  the  absence  of 
antibodies  in  sufficient  quantity  in  the  amount  of  serum 
used  to  fix  complement. 

4.  Give  all  results  in  detail  and  draw  conclusions. 

REFERENCES 

MOHLER,  J.  R.  and  EICHHORN,  A.:  The  diagnosis  of  glanders  by 
complement  fixation  test.  Bui.  136,  B.  A.  I.,  U.  S.  Dept.  Agr. 
(1911). 

SURFACE,  F.  M.:  The  diagnosis  of  infectious  abortion  in  cattle. 
Bui.  166,  Kentucky  Agr.  Expt.  Sta.  (1912). 

HADLEY,  F.  B.  and  BEACH,  B.  A.:  The  diagnosis  of  contagious  abor- 
tion in  cattle  by  means  of  the  complement  fixation  test.  Res. 
Bui.  24.  Wis.  Agr.  Expt.  Sta.  (1912). 

MOHLER,  JOHN  R.,  EICHHORN,  A.  and  BUCK,  J.  M.:  The  diagnosis 
of  dourine  by  complement  fixation.  Jour.  Agr.  Res.  Bui.,  Vol.  I 
(1913),  pp.  99-109. 

KOLMER:  Infection,  Immunity  and  Specific  Therapy  (1915),  pp.  146, 
164,  185,  316-501,  847-863. 

ZINSSER:    Infection  and  Resistance  (1914),  pp.  134-217. 


APPENDIX 
OUTLINE  FOR  THE  STUDY  OF  MICROBIOLOGY  * 


I.    MORPHOLOGICAL    AND    CULTURAL     MICRO- 
BIOLOGY 

A.  Morphology  and  Development. 

1.  Gross  anatomy. 

a.  Form. 

b.  Size. 

c.  Arrangement  or  grouping. 

d.  Multiplication. 

e.  Involution,  variability  and  mutation. 

2.  Histology  of  cell. 

a.  Wall  or  outer  membrane. 

b.  Capsule. 

c.  Protoplasm,  beaded  forms,  granules. 

d.  Nuclear  material. 

e.  Flagella  and  motion. 
/.  Spores. 

3.  Classifications  and  their  basic  features, 

B.  Cultural  Significance. 

1.  Media. 

a.  For  morphologic  and  developmental  studies. 

b.  For  cultural  effects. 

2.  Colonies. 

3.  Cultural  features. 

4.  Biochemical  features. 

*  Adapted  from  Marshall,  vide  43d  Annual  Report  of    Michigan 
State  Board  of  Agriculture. 

331 


332  APPENDIX 

C.  Staining  Values. 

1.  Demonstrations  of  parts  of  cell. 

2.  Identification  of  species. 

3.  Differentiation  of  species. 

D.  Determination  of  Microorganisms. 

1.  Methods  employed. 

2.  Differential  characteristics. 

II.    PHYSIOLOGIC  MICROBIOLOGY 

A.  Cell  Studies. 

1.  Composition  of  cell  contents. 

2.  Composition  of  cell  wall. 

3.  Physical  products  of  physiological  significance. 

a.  Heat. 
6.  Light. 

4.  Products  of  physiological  significance  of  which  little  is 

known, 
a.  Pigment. 
6.  Enzymes. 

c.  Aromatic  compounds. 

d.  Toxins. 

5.  Absorption  or  assimilation  of  foreign  bodies. 

6.  Chemotaxis. 
5.  Phototaxis. 

8.  Aerotaxis, 

9.  Plasmolysis. 
10.  Plasmoptysis. 

B.  Studies  in  Metabolism. 

1.  Elements  required  in  growth  of  microorganisms. 

2.  Respiration. 

3.  Nutrition. 

4.  Moisture. 

5.  Temperature  of  cultivation. 

6.  Conditions  of  media;  reaction,  composition,  etc. 


APPENDIX  333 

7.  Physiologic  test  media. 

8.  Identification  and  determination  of  species  of  micro- 

organisms by  means  of 

a.  Cultural  physiologic  methods. 

b.  Chemical  tests. 

c.  Physical  tests. 

d.  Biological  tests. 

9.  Enzymes. 

C.  Studies  in  Association. 

1.  Symbiosis. 

2.  Metabiosis. 

3.  Antibiosis. 

D.  Common  Fermentative  Changes  Produced  by  Micro- 

organisms. 

1.  Studies  in  enzymes. 
a.  Formation. 
Zymogen. 
Activator. 
Kinases. 
6.  Kinds. 

c.  Actions  (specificity)  and  materials  fermented. 

d.  Conditions  under  which  enzymes  act; 

(1)  Physical. 
Temperature. 

Radiation,  light  rays  (solar,  electric,  etc.), 
Rontgen  rays,  radium  rays  and  emana- 
tions. 

(2)  Chemical  and  physico-chemical. 
Activators;    kinases,    organic   acids,    bases, 

neutral  salts. 
Protective  agents. 
Paralysors  and  poisons. 
Concentration  of  solutions. 
Reaction  of  substrate. 
Extent  of  accumulated  products. 


334  APPENDIX 

2.  Products  manufactured  by  fermentation. 

a.  Necessary  and  limiting  conditions  of  production. 

b.  Most  favorable  conditions  of  production. 

c.  Methods  of  determination. 

Qualitative. 
Quantitative. 

d.  Constancy  and  variability  of  products. 

e.  Gradation  in  fermentation  changes. 

Intermediate  products. 
Ultimate  products. 

E.  Products  Significant  through  the  Intermediation  of  a 

Host. 

1.  Antigens.* 

a.  Cells. 

b.  Cell  products. 

Toxins,  diffusible  and  endotoxin. ' 

Bacterial  proteins. 

Enzymes. 

2.  Antibodies.* 

a.  Antitoxin. 

b.  Agglutinins. 

c.  Precipitins.        ^ 

d.  Opsonins. 

e.  Aggressins. 
/.   Cytolysins. 

g.  Anaphylactins. 

F.  Influencing  Agents  and  Their  Effects. 

1.  Light. 

a.  Direct. 
6.  Diffuse. 
c.  Special. 

*See  p.  164,  Kolmer's  Infection,  Immunity  and  Specific  Therapy 
(1915). 


APPENDIX  335 

d.  Phototropism. 

e.  Phototaxis. 

2.  Temperatures. 

a.  Heat. 

Direct  flame. 

Dry. 

Moist. 

Steam  under  pressure. 
6.  Cold. 

c.  Thermotaxis. 

d.  Thermotropism. 

3.  Electricity. 

4.  Desiccation. 

5.  Mechanical  pressure. 

6.  Mechanical  agitation. 

7.  Gravity. 

8.  Chemicals. 

a.  Chemotropism. 

b.  Chemotaxis. 

c.  Concentrated  solutions. 

d.  Antiseptics,  disinfectants, 

III.    HYGIENIC  MICROBIOLOGY 

A.  Communicable  Diseases  of 

1.  Man  and  animals. 

a.  Causal  agent  or  microorganism. 
6.  History  of  microorganism. 

c.  Vitality  or  persistency  of  microorganism. 

d.  Means  of  dissemination  and  avenues  of  infec- 

tion. 

e.  Distribution  of  microorganism  in  body. 
/.  Management  of  disease. 

g.  Prevention  of  disease. 

h.  Care  of  dead  from  communicable  diseases. 


336  APPENDIX 

B.  Surgical  Significance. 

1.  Wounds. 

2.  Abscesses. 

3.  Septicemia  and  pyemia. 

4.  Malignant  growths. 

5.  Operations. 

C.  Susceptibility  and  Immunity. 

1.  Natural. 

a.  Race. 

b.  Species. 

c.  Age. 

d.  Individual  idiosyncrasies. 

e.  Body  components. 

2.  Acquired,  active  or  passive. 

a.  Devitalization.  , 

b.  Hereditary  predisposition. 

c.  One  attack  of  disease. 

d.  Vaccines. 

e.  Bacterins. 
/.  Toxins. 

g.  Other  bacterial  products. 

D.  Serum  Therapy— Microbial  Therapeutics 

1.  Diagnostic  agents. 

a.  Tuberculin. 

b.  Mallein. 

c.  Bacterial  suspensions. 

d.  Diphtheria  toxin  (Schick). 

e.  Luetin. 

2.  Remedial  agents. 

a.  Antitoxins. 

b.  Serums. 

c.  Vaccines. 

d.  Bacterins. 


APPENDIX  337 

E.  Disinfection  and  Antisepsis. 

1.  Agents  employed. 

a.  Mode  of  action. 

2.  Determination  of  values,  phenol  coefficient. 

3.  Methods. 

F.  Sanitary  Studies. 

1.  Water  analysis. 

a.  Methods. 

b.  Interpretation  of  results. 

2.  Water  contamination  and  filtration. 

3.  Sewage  analysis. 

a.  Methods. 

b.  Interpretation  of  results. 

4.  Sewage' destruction. 

a.  Aerobic — filtration. 

b.  Anaerobic — septic  tank. 

c.  End  products. 

5.  Ventilation. 

a.  Currents  as  means  of  dissemination. 

b.  Filtration  and  washing  of  air. 

c.  Germ  content  of  air. 

d.  Methods  of  analysis. 

e.  Interpretation  of  results  of  analysis. 

6.  Foods. 

a.  Poisonous. 

b.  Infected. 

/ 
IV.    DAIRY 

A,  Milk  Supply. 

1.  Communicable  diseases  conveyed  through  milk. 

a.  Kinds  of  microorganisms. 

b.  Avenues  of  transmission. 

c.  Prevention. 


338  APPENDIX 

2.  Environment    of   animals   and    conditions   of   milk- 

ing. 

a.  Stabling. 

b.  Feeding. 

c.  Milker. 

d.  Utensils. 

3.  Bacterial  content  of  milk  in  udder. 

a.  Non-pathogenic  microorganisms. 

6.  Pathogenic  microorganisms  and  antibodies. 

c.  Conditions  of  growth  in  udder. 

d.  Abnormal  udders. 

4.  Bacterial  action  on  constituents  of  milk. 

a.  Proteins. 

b.  Butter  fat. 

c.  Lactose. 

d.  Mineral  constituents. 

5.  Analysis  Of  air  of  stables. 

a.  Before  cleaning. 

b.  Immediately  after  cleaning. 

c.  Before  feeding. 

d.  Immediately  after  feeding. 

e.  Analysis  of  out-door  air. 

6.  Determination  of  value  of  staining. 

7.  Determination  of  value  of  aeration. 

8.  Determination  of  value  of  cooling. 

a.  Simple  cooling. 

b.  Cooling  and  keeping  cool. 

c.  Cooling  and  warming,  then  cooling. 

9.  Cleansing  of  utensils. 

a.  Methods  and  their  values. 

b.  Water  analysis. 
10.  Milk  control. 

B.  Pigment  in  Milk  and  Cheese. 

1.  Kinds. 

2.  Character. 


APPENDIX  339 

3.  Condition  of  formation. 

4.  Control. 

C.  Fermentations  in  Milk,  Butter  and  Cheese. 

1.  Kinds. 

a.  Lactic. 

b.  Butyric. 

c.  Alcoholic. 

d.  Gaseous. 

e.  Peptic. 
/.   Rennet. 
g.  Ropy. 
h.  Soapy. 
i.   Taints. 

Bitter  flavor,  barn-yard,  tallowy. 
j.  Special. 

Kephir,  koumiss,  matzoon,  leben,  yoghurt,  etc. 
k.  Natural  enzymes  (galactase). 
I.  Antibody  formation  (agglutinins,  etc.). 

2.  Microorganism  involved. 

a.  Its  life  history. 

3.  Nature  of  fermentation. 

4.  Constituents  acted  upon. 

5.  Products. 

6.  Conditions  influencing  it. 

7.  Controlled  or  fostered. 

D.  Pasteurization  and  Sterilization. 

1.  Determination  of  significance  of  each. 

2.  Methods  employed. 

3.  Practical  utilization. 

E.  Starters. 

1.  Natural. 

a.  Sour  milk. 
6.  Sour  cream. 
c.  Buttermilk. 
(J,  Others, 


340  APPENDIX 

2.  Artificial. 

a.  Pure  cultures. 

b.  Mixed  cultures. 

3.  Value  determined. 

4.  Preparation. 

5.  Employment. 

6.  Constancy. 

7.  Influencing  conditions. 

8.  Facts  governing  amounts  to  employ. 

F.  Butter. 

1.  Microorganisms  present. 

2.  Microorganisms  compared  with  those  of  milk. 

3.  Environmental  condition  for  bacterial  life  changed. 

4.  Quality. 

a.  Influenced  by  pasteurization  of  the  cream. 

b.  Influenced  by  growth  of  microorganisms. 

c.  Factors  influencing  stability. 

d.  Methods  of  preservation. 

5.  Decomposition. 

a.  Products. 

b.  Factors  influencing. 

c.  Correlation   between   the   presence   of   certain 

groups  of  organisms  and  specific  flavors. 

6.  Significance  of  casein  and  buttermilk  in  butter. 

G.  Cheese. 

1.  Kinds    of    microorganisms    employed    in    different 

cheeses. 

2.  The  study  of  microorganisms  in  the  ripening  process. 

3.  Influence  of  microorganisms  on  aroma  and  flavor. 

4.  Keeping  values. 

H.  Preservatives. 

I.    Disinfectants  utilized. 


APPENDIX  341 

V.     SOIL 

A.  The  Making  of  Soil. 

1.  Microorganisms  in  soil. 

a.  Number  at  different   depths   and  in  different 

soils. 

b.  Kinds  at  different  depths  and  in  different  soils. 

c.  Character  of  microorganisms  found. 

d.  Rate  of  growth. 

2.  Disintegration  of  inorganic  material. 

3.  Decomposition  of  organic  material. 

a.  Celluloses. 

6.  Starches  and  sugars. 

c.  Proteins,  etc. 

4.  Action  of  iron  and  sulphur  bacteria. 

B.  Ammonification. 

C.  Nitrification — The  nitroso-  and  nitro-processes. 

1.  Conditions  influencing. 

a.  Physical. 

b.  Reaction. 

c.  Temperature. 

d.  Supply  of  oxygen. 

e.  Amount  of  organic  matter  present. 
/.  Moisture. 

D.  Denitrification. 

1.  Factors  influencing  the  loss  of  nitrogen. 

E.  Nitrogen  Fixation. 

1.  Symbiotic. 

2.  Nonsymbiotic  (aerobic  and  anaerobic). 


342  APPENDIX 

VI.     PLANT 

A.  Nitrogen  Accumulators. 

1.  Microorganism  involved. 

2.  Cultural  characteristics. 

3.  Formulation  of  nodules. 

4.  Character  of  nodules. 

5.  Conditions  under  which  they  form. 

6.  Determination  of  nitrogen  accumulations. 

7.  Significance  of  nodules. 

B.  Microbial  Diseases. 

1.  Kinds. 

2.  Microorganisms  found  as  causal  agents. 

3.  Cultural  characteristics. 

4.  Resistance  of  microorganisms. 

5.  Persistency. 

6.  Methods  of  treatment. 

7.  Pathology. 

C.  Microbial  Decomposition    of    Fruits,    Vegetables    and 

Other  Plant  Substances. 

1.  Nature. 

2.  Microorganism  studies. 

3.  Conditions  favoring. 

4.  Control. 

5.  Structural  changes.   . 

VII.     FERMENTATION 
A.  Factors  Controlling  Fermentations. 

1.  Presence  of  microorganism. 

2.  Purity  of  culture. 

3.  Vigor  of  cell. 

4.  Character  of  fermentable  material. 

5.  Air  supply. 


APPENDIX  343 

6.  Reaction  of  medium. 

7.  Temperature. 

8.  Concentration  of  fermentation  solutions. 

9.  Concentration  of  products  of  fermentation. 

B.  The  Production  of  Enzymes  by  Microorganisms. 

1.  Formation  of  enzyme  in  cell. 

2.  Its  secretion  by  the  cell. 

3.  Determinative  methods  for  study. 

4.  Environmental  influences. 

C.  The  Fermentations. 

General. 

1.  The  Enzymes. 

a.  Hydrolytic  of 

Carbohydrates  =  Carbohydrases. 

Cellulases. 

Hemicellulases. 

Glycogenases. 

Dextrinases. 

Inulinase. 

Saccharase. 

Lactase. 

Maltase. 

Trehalase. 

Raffinase. 

Amygdalase. 

Tannase. 

Pectase,  etc. 
Fats  =  Esterases. 

Lipases  of  natural  fats. 

Stearinases,  etc. 
Proteins  =  Proteinases. 

Peptases. 

Tryptases. 

Ereptases,  etc. 


344  APPENDIX 

b.  Producing  intramolecular  changes,  acting  on 

Carbohydrates,  to  form  alcohol  and  C02. 

Zymases  of  d-dextrose,  d-levulose,  etc. 
Carbohydrates  to  form  lactic  acid. 

Lactic  acid-bacteria  zymase. 
Acid  amides  =  amidases. 

Urease. 

c.  Oxidizing  =  oxidases. 

Alcoholase. 

Lactacidase. 

Acetacidase. 

Tyrosinase. 

Laccase. 

d.  Reducing  =  Reductases. 

Catalase. 

Peroxidase. 

Methylen  blue,  indigo  and  azolitmin  reduc- 

tase. 

Perhydridase. 
Sulphur  reductase. 
Nitrate  and  nitrite  reductase,  etc. 

e.  Coagulating. 

Caseinase. 
Parachymosin. 
Thrombase. 
Pectinase. 

2.  Materials  Acted  Upon. 

a.  Celluloses. 

6.  Starches. 

c.  Sugars. 

d.  Fats. 

e.  Proteins. 

/.   Organic  acids,  etc. 
g.  Alcohols. 

3.  Products  resulting. 


APPENDIX  345 

Special. 

1.  Alcoholic. 

a.  Beer  and  distilled  liquors. 

6.  Wine,  cider  and  other  fermented  fruit  juices. 

c.  Ginger  beer. 

d.  Koumiss,  etc. 

2.  Acetic  acid. 

a.  Vinegar. 

b.  Mashes. 

c.  Foods. 

I 

3.  Lactic  acid. 

a.  Milk. 

b.  Mashes. 

c.  Foods,  sauer  kraut,  brine  pickles,  etc. 

d.  Ensilage. 

4.  Butyric  acid. 

a.  Milk. 

b.  Mashes. 

c.  Foods. 

5.  Ammoniacal. 

a.  Urea,  uric  and  hippuric  acid. 

b.  Proteins  and  their  nitrogenous  fractions. 

6.  Proteolytic. 

a.  Proteins,  albumins. 

b.  Proteoses,  albumoses. 

c.  Peptones. 

d.  Peptids. 

e.  Amino-acids. 

/.   Amins  and  other  ammonia  derivatives. 

g.  Ptomains. 

h.  Leucomains. 

i.  Non-nitrogenous  organic  acids. 

7.   Alcohols. 

k.  Ammonia,  H2S,  and  other  gases. 


346  APPENDIX 

7.  Nitrification. 

8.  Denitrification. 

9.  Ammonification. 

VIII.    FOOD  AND  DRINK  PRESERVATION 

A.  Preservation  of  Foods. 

1.  Freezing. 

2.  Cold  storage. 

3.  Salting. 

4.  Drying,  evaporating  or  concentrating. 

5.  Smoking. 

6.  Corning. 

7.  Canning. 

8.  Chemical  preservatives  or  antiseptics. 

9.  Preserving. 

10.  Pressure. 

11.  Fermentations. 

B.  Preservation  of  Drinks. 

1.  Pasteurizing  and  sealing. 

2.  Cold  storage. 

3.  Chemical  preservatives. 

4.  Carbonating. 

5.  Filtration. 

6.  Fermentation. 


APPENDIX 


347 


CLASSIFICATION  OF  MIGULA  (MODIFIED) 


Order. 

Family. 

Genus. 

Species.         Variety- 

Streptococcus.  .  .    1 

division    in    1   1 

pyogenes 

plane,    no    fla-  1 

erysipelatus 

geUa 

Micrococcus.  •  •  •   1 

t>€'i/TCLQ^71AJLS 

division    in    2  1 

(  niivoitQ 

planes,  no  fla- 

\  u  u/r  t/  wo 

Py°9e™s  (albus 

geUa 

CoccacecB 

Sarcina  ] 

round 
forms 

division    in    3  1 
planes,  no  fla-  , 

lutea 

gella                   J 

1 

Planococcus  .-•..:.  1 

division    in    2 

agilis 

planes,  flagella  J 

I. 

Planosarcina  .  .  . 

EUBACTERIA 

(true 

division    in    3 
planes,  flagella   > 

'  mobiiis 

bacteria) 

t 

lactis  acidi 

Bacterium  

bulgaricum 

(straight  rods)    < 

aerogenes 

Suborder. 

(non-flagellate) 

abortus 

.  tuberculosis 

A. 

Haplobacte- 

Bci  ctarLcL  CBCB 

Bacillus 

fluorescens  lique- 
faciens 

TITiOB. 

(lower  bac- 

rod forms. 

(straight  rods) 
(flagellate) 

mycoides 
prodigiosus 
typhosus 

teria.) 

coli 

Pseudomonas.  .  .   ' 

(straight  or  ir- 

radicicola 

regular  rods, 

>  campestris 

polar  flagella) 

Spirosoma  ' 

comma  to  spi- 

> nasals 

SpirillaceoB 
curved 

ral  forms,  stiff, 
no  flagella. 

(comma)  or  • 

Microspira  

spiral 
forms. 

comma- 
shaped,  simple 

comma 
deneke 

curve,  general- 
ly    polar     fla- 

finkleri 

gella. 

348 


APPENDIX 


CLASSIFICATION  OF  MIGULA  (MODIFIED)— Continued 


Order.                      Family.                        Genus.                            Species. 

I. 

Spirillum  

EUBACTERIA 

cork  screw,  sev- 

(true bact.) 
Suborder. 

Spirillaceoe 
curved 

eral  turns,  non-      rubrum 
flexible    spiral, 

A. 

(comma)  or  • 

polar  flagella. 

Haplobacte- 

spiral 

Spirocheta  1 

rincK 

forms. 

flexible  spirals,   1  O5ema-er£ 

(lower  bac- 

motile, no  fla-  ) 

teria). 

gella.                   J 

See  pp.  10  and  56-62,  Marshall's  Microbiology. 

Chlamydothrix 

T 

Chlamydo- 

unbranched     threads,     uniform     in 

JL« 

EUBACTERIA 
(true   bacte- 

bacteriacece 
cylindrical 

diameter. 

Crenothrix 

ria). 
Suborder 

B. 

Trichobacte- 
rinoB  (higher 
bacteria). 

cells     in 
threads,  en-- 
sheathed; 
reproduc- 
tion by  mo- 
tile and  non- 
motile     go- 
nidia. 

unbranched    threads,   filaments    en- 
larged at  free  end. 
Phragmidiothrix 
branched  and  unbranched  filaments. 
Cell  division  in  3  planes. 
Cladothrix 
dichotomous  branching,  uniform  di- 

ameter. 

Beggiatoacece 

'  Thiothrix. 
threads,    non-motile   and   attached; 

cells      con- 
tain       sul- 

sheath; gonidia. 

phur   gran- 
ules. 

Beggiatoa 
no  sheath,  flat  cells,  motile  with  un- 

dulating membrane;  no  gonidia. 

II. 

{Thiocystis 

THIOBACTE- 

Rhodobacteri- 

Thiocapsa 

RIA      (sul- 

acece 

Thiosarcina 

phur    bac- 
teria). 

cells      con- 
tain bacte- 
riopurpurin, 

2      Lamprocystis 
3      Thiopedia 

sometimes 

(Amcebobacter 

sulphur 

Thiothece 

granules. 

Thiodictyon 

5  sub-fami- 

f Chromatiwn 

lies. 

5  \  Rhabdochromatium 

(  Thiospirillum 

APPENDIX  349 

SPECIAL  MEDIA 

Litmus  lactose  agar  for  demonstrating  acid  production 
of  microorganisms:  Prepared  the  same  as  ordinary  nutrient 
agar  (see  Exercise  9,  Part  I),  with  the  exception  that  1% 
lactose  and  2%  of  the  standard  azolitmin  solution  is  added 
just  after  filtration,  while  the  agar  is  still  hot,  and  well 
mixed  through  the  agar  before  tubing.  Sterilize  by  Tyndall 
method. 

Dextrose  calcium-carbonate  agar  for  showing  acid  for- 
mation by  microorganisms:  Prepared  the  same  as  ordinary 
nutrient  agar,  with  the  exception  that  1%  dextrose  and  1% 
CaCOs  are  added  to  the  hot  agar  just  after  filtration.  The 
added  chemicals  must  be  mixed  well  through  the  agar  and 
care  must  be  taken  during  tubing  that  the  CaCOs  remains 
in  homogeneous  suspension  throughout  the  medium.  Ster- 
ilize by  discontinuous  method. 

Sour  whey  for  determining  the  acid-destroying  power 
of  microorganisms:  Inoculate  sweet  milk  with  a  pure  active 
culture  of  Bad.  lactis  acidi  or  Bact.  bulgaricum  as  desired, 
and  place  at  about  30°  C.  Allow  the  maximum  acidity  to 
form,  cut  the  curd  and  heat  in  flowing  steam  for  twenty 
or  thirty  minutes.  Strain  through  clean  cheese-cloth  and 
allow  to  drain.  Filter  through  filter  paper.  If  clear  whey 
is  desired,  it  will  be  necessary  to  clear  the  medium  with 
egg  albumin. 

Butter  fat  for  demonstrating  fat  decomposition:  Melt 
butter  at  about  100°  C.  and  allow  the  casein  to  settle. 
Decant  the  clear  fat,  place  about  8  c.c.  in  sterile  test  tubes 
and  sterilize  by  the  intermittent  method. 

Other  kinds  of  fat  may  be  prepared  similarly. 

Fermented  agar  for  making  solid  synthetic  media  and 
for  testing  food  requirements  and  selective  powers  of  bac- 
teria: 1.  Place  a  weighed  amount  (three  parts)  of  agar  in 
a  large  bottle  and  to  this  add  200  parts  of  distilled  water. 

2.  Cover  the  mouth  of  the  bottle  with  parchment  paper 


350  APPENDIX 

or  several  layers  of  clean  cheese-cloth  and  allow  to  ferment 
spontaneously. 

3.  Change  the  water  in  the  bottle  occasionally,  replacing 
the  amount  of  water  removed,  with  the  same  amount  of 
clean,  distilled  water. 

4.  When  the  active  fermentation  (as  noted  by  the  evolu- 
tion of  gas)  has  ceased  entirely,  this  agar  should  be  placed 
in  an   agateware  pail,   counterpoised,   boiled   over  a  free 
flame  to  dissolve  the  agar,  counterpoised  and  any  loss  made 
up  with  distilled  water. 

5.  Place  in  tubes  or  flasks  as  desired  and  autoclav. 

Uschinsky's  asparagin  medium :    protein-free. 

Asparagin,  COOH  -  CH(NH2)  •  CH2  •  CO  -  NH2. .  3.4  gms. 

Sodium  chloride,  NaCl 5.0  gms. 

Magnesium  sulphate,  MgS04 0.2  gm. 

Calcium  chloride,  CaCl2 0.1  gm. 

Monobasic  acid  potassium  phosphate,  KH2PO4.  1 . 0  gm. 

Iron  sulphate,  FeSO4 Trace 

Distilled  water , , , 1000.0 c.c. 

Cohn's  solution:  inorganic  nitrogen  combined  with  an 
organic  acid. 

Monobasic  acid  potassium  phosphate,  KH2P04.  5 . 0  gms. 

Calcium  phosphate,  CasPCU 0.5  gm. 

Magnesium  sulphate,  MgSCU *. 5.0  gms. 

Ammonium  tartrate,  CH(OH)  •  COO  •  NH4 ....  10 . 0  gms. 

CH(OH)-COO-NH4 
Distilled  water 1000.0  c.c. 

Winogradski's  medium  for  nitrate  formation:  inorganic 
nitrogen  combined  with  inorganic  acid. 

Ammonium  sulphate,  (NH4)2S04 0.40  gm. 

Magnesium  sulphate,  MgSO4 0 . 05  gm. 

Dibasic  acid  potassium  phosphate,  K2HP04 .  .       0.10  gm. 


APPENDIX  351 

Sodium  carbonate,  Na2COs 0 . 60  gm. 

Calcium  chloride,  CaCk Trace 

Distilled  water • 1000 . 0  c.c. 

Winogradski's  medium  for  symbiotic  nitrogen-fixation: 

nitrogen-free. 

Dibasic  acid  potassium  phosphate,  K2HP04.  .  1.00  gm. 

Magnesium  sulphate,  MgS04 0 . 50  gm. 

Sodium  chloride,  NaCl 0.01  gm. 

Ferric  sulphate,Fe2  (864)3 - 0.01  gm. 

Manganese  sulphate,  MnS(>4 ,.  . .  0.01  gm. 

Dextrose,  CH2OH(CHOH)4CHO 20 . 00  gms. 

Distilled  water 1000.00  c.c. 

Gelatin  for  cultivating  phosphorescent  halophilic  or- 
ganisms: Prepared  as  ordinary  gelatin  with  the  addition 
of  3%  salt.  The  reaction  is  made  -20°. 

Fermented  cider  for  the  cultivation  of  acetic  bacteria: 
Inoculate  unfermented  cider  with  Sacch.  ellipsoideus  and 
allow  to  proceed  until  the  evolution  of  gas  ceases.  Filter, 
place  in  tubes  and  flasks  as  desired.  Pasteurize. 

MEDIA   FOR  SOIL   MICROBIOLOGY. 

Soil  extract:  1.  Boil  1  kg.  of  good  rich  garden  soil 
with  2  liters  of  tap  water  for  two  hours  over  the  free  flame. 

2.  Pour  off  the  turbid  liquid,  mix  some    talc- and  filter 
through  a  double  filter  paper.     If  the  first  filtrate  is  turbid 
refilter  through  the  same  paper. 

3.  Make  up  to  800  c.c.  with  tap  water. 

4.  Place  in  tubes  or  flasks  as  desired  and  autoclav. 
Soil  extract  agar  is  prepared  by  adding  1.5%  washed 

agar  to  the  soil  extract  prepared  as  above. 

Soil  may  be  plated  either  in  soil  extract  agar  (or  other 
special  agar)  or  in  ordinary  agar,  gelatin,  etc.  On  account  of 
the  diversity  of  the  requirements  of  the  various  species  of 


352  APPENDIX 

microorganisms  in  soil,  no  one  medium  will  suffice  for  the 
cultivation  of  all  species.  Emphasis  is  therefore  not  laid 
on  any  particular  medium  for  plating  soils. 

Omeliansky's  medium  for  anaerobic  cellulose  fer- 
mentation : 

Filter  paper  (in  strips).      Cotton,  straw,  or 

starch  may  be  substituted  for  filter  paper .  2.0  gms. 

CaC03.. 20.0  gms. 

K2HPO4. . 1.0  gm. 

MgS04 0.5  gm. 

(NH4)2SO4 1 .0  gm. 

NaCl Trace 

Distilled  water 1000.0  c.c. 

Method.  1.  Introduce  substances  in  order  named 
into  1000  c.c.  distilled  water. 

2.  Stir  to  dissolve  all  soluble  substances  and  tube  while 
insoluble  substances  are  in  homogeneous  suspension,  plac- 
ing about  10  c.c.  in  each  tube. 

3.  Sterilize  in  autoclav. 

Media  for  studying  urea  decomposition:  Urea  broth, 
gelatin  and  agar  are  generally  prepared  by  adding  1%  to 
2%  urea  to  the  ordinary  media.  This  medium  favors  the 
growth  of  B.  coli,  B.  proteus,  B.  erythrogenes,  etc. 

Ordinary  media  to  which  10%  urea  has  been  added  favors 
the  growth  of  B.  pasteurii,  a  spore-producing  bacterium. 

Urea  gelatin  and  agar  may  be  prepared  by  adding  1 
c.c.  of  a  15%  aqueous  solution  of  urea  to  each  tube  of  the 
ordinary  media  after  sterilization,  and  then  heating  the 
tubes  again.  This  is  the  method  preferred  because  the 
addition  of  urea  reduces  the  solidifying  power  of  the  gelatin. 
A  small  amount  of  urea  is  converted  into  ammonia  by 
heating  in  the  steam,  but  this  has  little  influence  on  the 
results  obtained  in  the  experiment.  Heating  in  the  auto- 
clav is  to  be  avoided! 


APPENDIX  353 

Albuminoid-free    culture    solutions   for    studying   urea 
decomposition : 

I.  Soil  extract 100  c.c. 

K2HP04 O.OSgm. 

Urea 5 . 00  gms. 

II.  Sohngen's  solution. 

Tap  water 100.00  c.c. 

Urea 5 . 00  gms. 

K2HPO4. 0.05gm. 

Ammonium   or  calcium  malate, 

or,  calcium  citrate  or  tartrate.      0. 50  to  1.00  gm. 

B.  pasteurii  will  not  grow  in  these  solutions  as  it  requires 
the  presence  of  albuminoids  in  the  medium. 

Solutions  for  cultivating  nitrifying  bacteria: 

I.  Distilled  water 1000.0  c.c. 

(NH4)2SO4 1.0  gm. 

K2HP04 1.0  gm. 

MgS04.... 0.5gm. 

NaCl 2.0  gms. 

FeS04 0.4gm. 

Add  basic  MgCO3  after  sterilizing. 
This  solution  is  adapted  for  relatively  increasing  the 
nitrite  bacteria. 

H.  Distilled  water 1000.0  c.c. 

NaN02 1.0  gm. 

K2HPO4 0.5gm. 

MgSO4 0.3gm. 

NaCl 0.5gm. 

Na2CO3 0.3gm. 

This  solution  causes  a  greater  relative  increase  in  the 
nitrate  producers. 


354  APPENDIX 

III.  The  same  as  solution  I,  but  instead  of  MgC03 
CaCOs  is  added  after  sterilizing.  This  solution  stimulates 
the  simultaneous  growth  of  both  organisms,  as  in  nature. 

Culture  solutions  for  denitrification  studies.  Nitrate 
broth  or  agar.  Add  1  c.c.  of  a  1%  solution  of  sodium  or 
potassium  nitrate  to  tubes  of  ordinary  broth  or  agar  (melted), 
mix  well  and  re-sterilize. 

Giltay's  solution. 

KH2PO4. 2.0  gms. 

MgS04.  .    2.0  gms. 

KNO3-.... 1.0  gm. 

CaCl2 0.2  gm. 

Fe2Cle  solution 2.0  drops 

Citric  acid 5.0  gms. 

Method.  1.  Dissolve  the  above  substances  in  800  c.c. 
of  distilled  water  (solution  I). 

2.  Add  a  few  drops  of  phenolphthalein  -and,  using  a 
pipette,  drop  in  just  enough  10%  NaOH  to  turn  the  solution 
a  faint  pink. 

3.  Dissolve  10  gms.  dextrose  in  200  c.c.  of  distilled  water 
(solution  II). 

4.  Mix  solutions  I  and  II  very  thoroughly. 

5.  Sterilize  in  the  autoclav  at  15  Ibs.  pressure   for  ten 
minutes.     (Lipman  and  Brown.) 

Giltay's  agar  is  prepared  by  adding  1.5%  washed  agar 
to  the  above  solution.  Boil  until  dissolved.  Filter  through 
absorbent  cotton.  Sterilize  in  autoclav. 

Mannit  solution  for  nitrogen-fixing  organisms. 

Mannit 15.0  gms. 

K2HP04 0.2gm. 

MgSO4 0.2gm. 

NaCl 0.2gm. 

CaSO4 0.1  gm. 

CaC03 , 5.0  gms. 

10%  FeCl6  solution 1.0  drop 


APPENDIX  355 

Method.  1.  Add  -the  above  chemicals  to  1000  c.c. 
distilled  water. 

2.  Titrate   using  phenolphthalein   and  neutralize  using 
normal  NaOH. 

Do  not  filter.  The  presence  of  CaCOs  offers  an  additional 
means  of  isolating  Azotobacter,  as  these  organisms  are  found 
in  soil  in  much  greater  numbers  around  the  particles  of 
calcium  carbonate. 

3.  Sterilize  at  120°  C.  (autoclav),  for  ten  minutes. 
Mannit  agar  is  prepared  by  adding  1.5%  washed  agar 

to  the  above  solution,  boiling  until  the  agar  is  wholly  dis- 
solved and  sterilizing  as  above.     Do  not  filter. 

Nitrogen-free  ash  agar  for  cultivation  of  Ps.  radicicola. 

1.  Stir  5  gins,  of  wood  ashes  (beech,  elm,  maple)  into 
1000  c.c.   distilled  water  for  two  to  three  minutes  only. 
Filter. 

2.  Add  1%  washed  agar. 

3.  Heat  in  steam  for  thirty  minutes. 

4.  Then  add  1%  commercial  saccharose. 

5.  Boil  five  minutes  over  a  free  flame. 

6.  Strain  while  hot  through  several  thicknesses  of  clean 
cheese-cloth.     This  may  be  filtered  if  desired. 

7.  For    Exercise    9,    Soil    Microbiology,    tube,    placing 
about  6  cm.  of  agar  in  the  large  test  tubes  with  foot,  the 
rest  in  ordinary  test  tubes.     Sterilize.     (Tyndall  method.) 

Nitrogen-free  solution  may  be  prepared  as  above, 
omitting  the  agar. 

Congo-red  agar  for  differentiating  Ps.  radicicola  from  Bact. 
tumefaciens: 

Distilled  water 1000.00  c.c. 

Saccharose 10.0  gms. ' 

K2HP04 1.0  gm. 

MgSO4 0.2gm. 

Washed  agar 15.0  gms. 

Congo-red 0.1  gm. 


356  APPENDIX 

Solution  for  sulphate  reduction: 

Tap  water 1000.0  c.c. 

K2HPO4 7. 0.5gm. 

Sodium  lactate 5.0  gms. 

Asparagin 1.0  gm. 

MgSO4 1.0  gm. 

A  few  drops  of  FeSCU  solution.  Sterilize  in  the  auto- 
clav. 

WATER  ANALYSIS  MEDIA 

Culture  media  for  standard  bacteriological  water  anal- 
ysis must  contain  ingredients  of  a  special  nature. 

Ingredients.     1.  Distilled  water  in  place  of  tap  water. 

2.  Infusion  of  fresh  lean  meat  instead  of  meat  extract. 

3.  Witte's  peptone  (dry,  from  meat) . 

4.  No  salt. 

5.  Gelatin  of  the  best  French  brand  and  as  free  as  pos- 
sible from  acids  and  other  impurities. 

6.  Commercial  agar  of  as  high  a  grade  of  purity  as 
possible.     Agar  may  be  purified  by  washing. 

7.  Dextrose,  lactose,   saccharose,  etc.,  of  sugar  media, 
chemically  pure. 

8.  A    1%   aqueous   solution   of   Kahlbaum's   azolitmin 
may  be  used  in  place  of  litmus. 

Sterilization.  Sterilize  media  in  the  autoclav  at 
120°  C.  (15  Ibs.  pressure)  for  fifteen  minutes.  A  shorter 
period  than  this  may  result  in  incomplete  sterilization,  a 
longer  period  will  probably  result  in  inversion  and  cara- 
melization  of  the  sugars  and  in  lowering  the  melting-point 
of  the  gelatin.  Have  the  sterilizer  hot  when  the  medium  is 
inserted  so  that  heating  to  the  point  of  sterilization  will  be 
accomplished  as  quickly  as  possible;  cool  rapidly  upon  re- 
moving from  the  autoclav. 

The  Tyndall  (intermittent)  method  may  be  employed, 
heating  for  thirty  minutes  on  three  successive  days, 


APPENDIX  357 

Reaction.    Phenolphthalein  is  used  as  indicator. 

Titrate  media  while  hot  with  N/20  NaOH  and  adjust 
the  reaction  if  necessary.  All  media  should  have  a  +10° 
reaction  Fuller's  scale  unless  otherwise  stated  in  the  direc- 
tions. 

Distribution  of  Work.  It  may  be  desirable  to  have 
students  work  in  groups  in  preparing  media.  The  fol- 
lowing plan  has  worked  satisfactorily: 

Students  may  work  in  groups  of  five,  one  of  the  groups 
preparing  a  sufficient  quantity  of  medium  for  himself  and_the 
other  four  members  of  the  group,  dividing  the  work  up  as 
follows : 

One  student  prepare  agar  shakes  and  litmus  milk. 

One  student  prepare  gelatin. 

One-  student  prepare  litmus  lactose  agar. 

One  student  prepare  litmus  lactose  bile. 

One  student  prepare  Dunham's  solution  and  nitrate 
peptone  solution.  , 

In  this  arrangement  each  student  must  furnish  the  re- 
spective sterile  glassware  sufficient  for  containing  the  various 
necessary  media,  to  the  student  preparing  each  medium. 

Each  student  of  the  group  must  so  plan  his  work  that  the 
medium  he  prepares  will  be  finished,  sterilized  and  ready  for 
use  at  the  same  time  as  those  of  the  remaining  members  of 
his  group. 

Media.     Litmus  lactose  agar  shake. 

1.  2%  washed  agar. 
2%  peptone. 
2%  lactose. 

2%  azolitmin. 

500  c.c.  meat  infusion. 

500  c.c.  distilled  water. 

Method.  1.  Strain  the  meat  infusion  through  a  piece 
of  clean  cheese-cloth. 

2.  Place  the  washed  agar  in  the  distilled  water,  weigh, 


358  APPENDIX 

digest  over  a  free  flame,  weigh  again  and  make  up  any  loss 
with  distilled  water. 

3.  To  the  hot  agar  add  the  peptone  and  lactose  and  mix 
until  dissolved;  then  add  the  strained  meat  infusion. 

4.  Titrate  and  adjust  the  reaction  to  0°. 

5.  Add  the  azolitmin,  boil  up  over  the  free  flame  and 
place  about  100  c.c.  in  sterile  250  c.c.  Florence  flasks. 

Each  student  will  need  four  litmus  lactose  agar  shakes. 

II.  Litmus  lactose  agar.     (To  be  used  in  tubes  for  plat- 
ing only.) 

1.5%  agar. 

1 . 0%  peptone. 

1.0%  lactose. 

2 . 0%  azolitmin  solution. 

500  c.c.  meat  infusion. 

500  c.c.  distilled  water. 

Method.  1.  This  agar  is  prepared  as  ordinary  nutrient 
agar  making  the  reaction  +10°,  adding  the  lactose  and  azo- 
litmin just  before  tubing. 

2.  Tube  and  sterilize  by  the  Tyndall  method. 

Each  student  will  need  at  least  forty  tubes  of  litmus  lactose 
agar. 

III.  Gelatin. 

15%  gelatin. 

1%  peptone. 
500  c.c.  meat  infusion. 
500  c.c.  distilled  water. 

Method.  Prepare,  tube  and  sterilize  as  for  ordinary 
gelatin. 

Salt  is  omitted.     Reaction  +10°. 

Each  student  will  need  forty  or  fifty  tubes  of  salt-free  gela- 
tin. 

IV.  Sugar-free   broths   and   sugar  broths.     (Neutral   red 
dextrose  broth.) 


APPENDIX  359 

Method.  1.  Heavily  inoculate  a  tube  of  sterile  broth 
with  B.  coli  and  incubate  at  37°  C. 

2.  Soak  1  Ib.  finely  chopped  lean  beef  in  1000  c.c.  dis- 
tilled water  over  night  (twenty-four  hours). 

3.  Strain  out  the  meat  juice  and  make  up  to  1000  c.c. 
with  distilled  water. 

4.  Pour    the    entire  contents  of    the    twenty-four-hour 
broth  culture  of  B.  coli  into  the  meat  juice  and 

5.  Incubate  at  37°  C.  for  twelve  to  sixteen  hours,  not 
longer.      B.  coli  uses   the   fermentable   substances,   inosite 
(muscle  sugar),  dextrose,  etc.,  as  food,  leaving  the  meat 
juice  free  from  fermentable  substances.     //  this  action  is 
allowed  to  proceed  too  long,  poisonous  decomposition  products 
of  the^  proteins  are  formed  which  will  inhibit  the  growth  of 
other  microorganisms. 

6.  Mix  the  peptone  (1%)  into  a  thin  paste  with  as  little 
water  '  as  possible  and  add  to  the  twelve  or  sixteen-hour 
culture  of  B.  coli  in  the  meat  juice. 

7.  Heat  in  the   autoclav  for  twenty  minutes  or  in  the 
steam  for  one  hour. 

8.  Titrate  and  make  neutral  to  phenolphthalein. 

9.  Boil  over  a  free  flame  for  three  to  five  minutes. 

10.  Add  1%  dextrose  and  10  c.c.  of  0.5%  solution  of 
neutral  red  and  stir  until  sugar  is  dissolved. 

11.  Filter  until  clear. 

12.  Fill  ten  fermentation  tubes  for  each  student. 

13.  Sterilize  in  autoclav  or  in  flowing  steam. 

14.  Other  sugar  broths  are  prepared  by  adding  instead 
of  dextrose,  1%  of  the  sugar  desired. 

Practically  all  sugar-fermenting  organisms  will  ferment 
monosaccharides  such  as  dextrose;  comparatively  few  will 
ferment  the  disaccharides  lactose,  saccharose,  etc.  B. 
coli  will  ferment  all  three  sugars  to  a  greater  or  less  extent. 
Bacteria  of  the  typhoid  group  ferment  none  of  the  three  and 
those  belonging  to  the  paratyphoid  group  ferment  dex- 
trose but  not  lactose,  therefore  the  use  of  lactose  in  culture 


360  APPENDIX 

media  will  inhibit  to  a  great  extent  the  growth  of  the  last 
two  groups  and  favor  the  development  of  the  organisms 
of  the  B.  coli  group.  This  group  is  by  far  the  largest, 
occurs  most  often  and  in  greatest  numbers  in  sewage  and 
like  'material,  therefore  tests  for  this  group  are  used  as  indi- 
cation of  the  presence  of  intestinal  organisms  in  the  material 
(water  in  this  case)  to  be  examined. 

V.  Litmus  lactose  bile  salt  medium. 

Bile  salts  are  invaluable  for  certain  media  used  for 
water  analysis  as  they  inhibit  organisms  of  practically  all 
but  the  intestinal  type. 

20  gms.  peptone. 
5  gms.  sodium  taurocholate. 
10  gms.  lactose. 
20  c.c.  2%  azolitmin  solution. 
1000  c.c.  distilled  water. 

Method.  1.  Dissolve  the  bile  salt  and  peptone  in  the 
water  and  boil. 

2.  Add  the  lactose  and  sufficient  azolitmin  to  give  a 
distinct  purple  tint. 

3.  Filter,  fill  into  fermentation  tubes  and  sterilize  by 
intermittent  method. 

Each  student  needs  four  litmus  lactose  bile  salt  fermentation 
tubes. 

VI.  Esculin  bile  solution  for  B.  coli  test. 

10 . 0  gms.  peptone. 
5 . 0  gms.  sodium  taurocholate. 
0.1  gm.    esculin. 
0 . 5  gms.  soluble  iron  citrate. 
1000.0  c.c.  distilled  water. 

Method.  1.  Dissolve  the  ingredients  in  the  order  given, 
clear  with  egg  albumen,  tube  and  sterilize  (see  Prescott  and 
Winslow's  Elements  of  Water  Bacteriology,  3d  Ed.,  p.  279). 
This  solution  has  a  blue  fluorescence. 


APPENDIX  361 

VII.  Dunham's  solution;  twenty-five  tubes  for  each  stu- 
dent.    (See  p.  43.) 

VIII.  Nitrate    peptone  solution;     twenty-five  tubes    for 
each  student.     (See  p.  44.) 

IX.  Litmus  milk;    twenty-five  tubes  for  each  student. 
(See  p.  25.) 

EXPLANATION   OF   TABLE   ON   PAGES  362-363 

Method.  B.  coli-  and  B.  cholerce  suis-like  organisms: 
Place  5  c.c.  of  suspected  water  in  each  dextrose  and  liver 
broth  fermentation  tube  and  50  to  100  c.c.  in  a  litmus  lactose 
agar  flask.  Incubate  at  37°  C.  If  gas  appears  in  time  of 
three  days,  make  plating  on  Conrad-Drigalski's  agar  *  from 
one  showing  most  of  gas  production.  Isolate  different 
colonies  on  agar  slants.  From  the  growth  on  the  agar 
slants  inoculate  different  media  to  subgroup  the  organisms 
and  consequently  to  identify  them. 

B.  typhosus:  Hoffman  and  Fiske  enrichment  medium. 
Add  to  the  suspected  water  1.0%  of  nutrose;  0.5%  of 
caffein;  0.001%  of  crystal  violet.  Incubate  at  37°  C. 
for  not  more  than  twelve  to  thirteen  hours.  Make  Endo 
or  Conradi-Drigalski  agar  plates.  Isolate  bluish  colonies, 
transferring  to  agar  slant,  and  identify.  The  Widal  reac- 
tion should  be  used  for  the  confirmatory  test. 

(Data  on  pages  362-363  collected  by  0.  M.  Gruzit.) 

*  Other  media  for  bacteriological  water  analyses  will  be  found  in 
the  1915  edition  of  the  "  Standard  Methods  for  the  Examination  of 
Water  and  Sewage  "  published  by  the  American  Public  Health  Associ- 
tion,  pp.  124-137. 

This  publication  is  the  standard  work;  references  to  special  phases 
will  be  found  in  the  bibliography  following  each  chapter. 


362 


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364  APPENDIX 

COMMON   DISINFECTANTS 

Mercuric  chloride :     (HgCl2)  White  crystals. 

Synonyms:  mercury  bichloride,  corrosive  sublimate, 
bichloride  of  mercury. 

The  stock  solution  (40%  HgCk  in  HC1)  is  prepared  by 
mixing  1  part  mercuric  chloride  with  2.5  parts  commercial 
hydrochloric  acid.  This  dissolves  readily  and  aqueous 
solutions  of  any  desired  dilution  may  be  made  from  it  much 
more  quickly  than  by  the  use  of  the  salt. 

The  pure  salt  is  soluble  in  16  parts  of  cold  water  and  3 
parts  of  boiling  water. 

Mercuric  chloride  1  :  1000,  the  solution  commonly  used 
in  the  laboratory  for  disinfecting  purposes,  is  prepared  by 
adding  to  2.5  c.c.  of  the  stock  solution,  sufficient  dis- 
tilled water  to  make  1000  c.c.  of  solution. 

As  a  germicide,  mercuric  chloride  acts  in  solution  by 
combining  chemically  with  the  protein  of  the  microorganisms. 
Therefore  its  efficiency  varies  in  inverse  proportion  to  the 
amount  of  dead  organic  matter  present. 

Mercuric  chloride  is  exceedingly  corrosive  as  is  also 
the  acid  in  which  it  is  originally  dissolved;  therefore  it 
should  not  be  placed  in  metal  containers  or  agateware 
pails,  cups,  etc.,  if  the  enamel  is  chipped  sufficiently  to 
expose  the  metal. 

Remember  that  mercuric  chloride  is  a  DEADLY  POISON! 
Great  care  must  be  exercised  in  properly  labelling  all  bottles, 
etc.,  containing  it. 

Phenol:  (CeHsOH)  long  colorless  crystals  that  become 
pink  upon  exposure  to  light  and  air. 

Synonyms:  Carbolic  acid,  phenic  or  phenylic  acid, 
phenyl  hydrate,  hydroxybenzene  (or  -ol). 

The  stock  solution  (95%  phenol)  is  prepared  by  adding 
1  part  of  water  to  19  parts  (by  weight)  of  crystalline  phenol. 
Solution  may  be  hastened  by  placing  the  vessel  containing 
the  crystals  in  a  dish  of  warm  water, 


APPENDIX  365 

Note.  When  making  up  the  stock  solution  or  dilutions  from  the 
stock  solution  always  have  a  bottle  of  ethyl  alcohol  at  hand  as  a  remedy 
for  burns  caused  by  phenol.  5%  phenol  is  prepared  by  adding  one 
part  of  95%  phenol  to  nineteen  parts  of  distilled  water. 

Its  value  as  a  disinfectant  is  increased  by  the  fact  that 
it  acts  in  the  presence  of  albuminous  substances.  It  does 
not  corrode  metals  or  destroy  fabrics  in  a  5%  solution. 

Liquor  cresolis  compositus,  U.  S.  P. 

Cresol 500  gms. 

Linseed  oil 350  gms. 

Potassium  hydroxide 80  gms. 

Water,  a  sufficient  quantity  to  make 1000  gms. 

Dissolve  the  potassium  hydroxide  in  50  gms.  of  water 
in  a  tared  dish,  add  the  linseed  oil,  and  mix  thoroughly. 
Then  add  the  cresol  and  stir  until  a  clear  solution  is  pro- 
duced. Finally  add  sufficient  water  to  make  the  finished 
product  weigh  1000  gms.,  or  more  briefly:  mix  equal  parts 
by  weight  of  cresol  and  linseed  oil-potash  soap  (Sapo  mollis, 
U.  S.  P.). 

This  mixture  is  a  thick,  dark,  amber-colored  fluid  which 
mixes  readily  with  water  in  all  proportions  to  form  a  clear, 
soapy  solution.  A  3%  or  4%  solution  will  accomplish  the 
same  results  as  5%  phenol.  It  is  not  interfered  with  by 
albuminous  substances  and  does  not  destroy  metals  or 
fabrics. 

Tincture  of  iodin,  U.  S.  P. 

lodin '. . .       70  gms. 

Potassium  iodid 50  gms. 

Alcohol,  sufficient  to  make 1000  c.c. 

Triturate  the  iodin  and  potassium  iodid  in  a  mortar 
to  a  coarse  powder  and  transfer  at  once  to  a  graduated 
flask.  Rinse  the  mortar  with  several  successive  portions 


366  APPENDIX 

of  alcohol  and  pour  the  rinsings  in  the  bottle;  then  add 
alcohol,  shaking  occasionally  until  the  iodin  and  potassium 
iodid  are  all  dissolved  and  the  finished  tincture  measures 
1000  c.c. 

SOLUTIONS  FOR  CLEANING  GLASSWARE 
Chromic  acid  cleaning  solution  for  cleaning  glassware: 

Potassium  or  sodium  dichromate 60  gms. 

Commercial  sulphuric  acid 60  c.c. 

Water 1000  c.c. 

Prepare  in  a  flask  resistant  to  heat,  never  in  a  heavy 
glass  jar. 

Add  the  potassium  dichromate  to  about  500  c.c.  water; 
shake  well  and  add  the  sulphuric  acid  gradually,  continually 
shaking  with  a  rotary  motion.  The  remaining  water  may 
then  be  added.  The  potassium  dichromate  should  be  all 
dissolved  before  using  the  solution. 

This  solution  may  be  used  repeatedly  until  oxidized 
to  a  dark  green  color.  Heat  will  hasten  its  action. 

Chromic  acid  cleaning  solution  is  especially  valuable 
for  removing  traces  of  oxidizable  organic  matter  and  neu- 
tralizing '  any  free  alkali  adhering  to  glassware.  However, 
effort  should  be  made  to  previously  remove  as  much  extrane- 
ous matter  as  possible  with  water  and  a  suitable  brush 
before  treating  with  this  solution.  This  will  economize 
time. 

Caution.  This  solution  contains  sufficient  sulphuric  acid 
to  destroy  fabrics,  bristles  of  brushes,  and  corrodes  metal 
quickly.  For  this  reason  neither  cloth  nor  brushes  should 
be  used  as  an  immediate  aid  to  this  cleaning  agent,  nor 
should  this  solution  be  placed  in  agateware  utensils  if  the 
enamel  is  chipped,  exposing  the  metal. 

If  this  solution  is  used  cold,  leave  the  glassware  contain-^ 
ing  it,  over  night  on  top  of  desk,  never  inside  of  desk. 


APPENDIX  367 

Sodium  hydroxide  solution  for  cleaning  glassware  and 

absorbing  C02 : 

Sodium  hydroxide,  sticks 100  gms. 

Water 1000  c.c. 

Use  only  once  if  the  glassware  is  very  dirty. 

This  solution  is  invaluable  for  cleaning  greasy  flasks, 
pipettes,  etc. 

Caution.  This  solution  should  not  be  left  in  contact 
with  any  glassware  longer  than  thirty  minutes  as  it  etches 
the  glass. 

A  sodium  hydroxide  solution  of  this  strength  is  very 
corrosive,  attacking  cloth,  laboratory  desk  tops,  etc.,  and, 
therefore,  should  be  wiped  up  immediately  if  spilled. 

This  strength  may  also  be  employed  to  absorb  C02 
in  fermentation  tube  cultures  of  gas-producing  organisms. 

STANDARD  SOLUTIONS 

A.  Preparation  of  N/10   Na2CO3  from  titration  against 
which  normal  acid  is  prepared. 

1.  Dry  finely  powdered  chemically  pure   Na2CO,3   in  a 
drying  oven  at  105°  C.  for  two  hours. 

2.  Weigh  out  carefully  and  as  accurately  as  possible 
5.3  gms.  of  the  dried  salt. 

3.  Dissolve   in   distilled  water  which   has   been  boiled 
previously  to  expel  C02  and  then  cooled. 

4.  Make  up  solution  to  one  liter,  using  a   calibrated 
volumetric  flask  and  observing  temperature  for  which  the 
flask  was  calibrated. 

5.  Keep  this  N/10  solution  of  Na2COs  in  a  stoppered 
bottle.     It  should  be  used  as  soon  as  possible  after  prepara- 
tion, as  the  Na2COs  acts  upon  the  glass  and  thus  deterio- 
rates. 

B.  Preparation  of  N/l,  N/10  and  N/20  HC1. 

1.  Measure  out  77.5  c.c.  HC1  (sp.gr.  1.20)  or  138  c.c. 


368  APPENDIX 

HC1  (sp.gr.  1.12)  and  make  up  to  one  liter  with  distilled 
water.  This  makes  a  solution  just  a  little  stronger  than 
normal. 

2.  To  determine  its  exact  strength,  titrate  5  c.c.  with 
N/10  Na2COs,  using  phenolphthalein  as  the  indicator. 

3.  Rim  check  determinations,  which  should  check  within 
one-  or  two-tenths  of  a  cubic  centimeter. 

4.  From  results,  calculate  by  proportion  how  much  a 
liter  of  the  solution  should  be  diluted  to  make  it  N/l.     e.g. : 

5  c.c.  HC1  was  neutralized  by  55  c.c.  N/10  Na2C03 

.'.     HC1  is  N/l.l 
By  proportion: 

N/l  :  N/l.l::  1000  :x 

a:  =  1100 

Hence  each  liter  of  the  HC1  solution  should  be  diluted  to 
1100  c.c.  with  distilled  water  to  make  a  N/l  solution  of 
HC1. 

5.  N/10  and  N/20  solutions  of   HC1  can  be   made  by 
making  the  proper  dilutions.     Always  use  calibrated  flasks 
and  burettes  when  making  these  dilutions. 

C.  Preparation  of  N/l  and  N/20  NaOH. 

1.  Weigh  out  roughly  41  gms.  of  chemically  pure  NaOH. 

2.  Dissolve  in  distilled  water,  which  has  been  boiled 
to  expel  CO2  and  then  cooled. 

3.  Make  up  to  one  liter,  using  a  calibrated  volumetric 
flask  and  observing  the  temperature  for  which  it  was  cali- 
brated.  This  makes  a  solution  a  little  stronger  than  normal. 

4.  Determine  its  exact  strength  by  titration  with  N/10 
HC1. 

5.  Proceed  from  this  point  as  in  the   preparation  of 
N/l  HC1. 

6.  N/10  and  N/20  solutions  can  be  made  from  the  N/l 
solution  as  in  the  preparation  of  N/10  and  N/20  HC1. 


APPENDIX  369 

INDICATORS 
Phenolphthalein,  indicator  for  titration: 

Phenolphthalein 0.5  gm. 

50%  alcohol  (neutral) 100.0  c.c. 

A  drop  of  a  weak  solution  of  alkali  should  produce  per- 
manent pink  color  when  added  to  a  small  amount  of  this 
solution.  Phenolphthalein  is  colorless  in  the  presence  of 
acid. 

Kahlbaum's  azolitmin  solution:  Dissolve  2.5  gms.  of 
Kahlbaum's  azolitmin  in  100  c.c.  distilled  water  by  heating 
in  steam  for  half  an  hour.  Filter  (this  will  filter  much 
more  readily  if  allowed  to  settle  for  some  time;  decant 
upon  the  filter).  Sterilize  by  heating  fifteen  minutes  each 
day  on  three  successive  days.  Sterilization  is  necessary, 
otherwise  molds  and  other  microorganisms  will  grow  on  the 
organic  material  present,  often  changing  the  reaction. 

A  solution  of  litmus  or  azolitmin  is  often  added  to  sugar 
and  other  media  before  sterilization  for  the  purpose  of 
detecting  microorganisms  which  produce  a  change  in  the 
reaction  of  the  media. 

Litmus  is  a  mixture  of  dyes  obtained  from  the  lichens 
Roccella  and  Lecanom  by  allowing  them  to  ferment  after 
the  addition  of  ammonia  and  potassium  carbonate.  When 
the  mass  has  assumed  a  deep  blue  color,  the  liquid  is  pressed 
out,  absorbed  by  chalk  or  gypsum,  and  dried. 

Merck's  purified  litmus,  often  used  in  bacteriological 
work,  is  made  from  commercial  litmus  solution  by  freeing 
it  from  the,  red  pigment  orcin,  and  drying  without  absorb- 
ing it  by  means  of  chalk  or  gypsum. 

Azolitmin  is  a  purified  pigment  from  litmus. 


370  APPENDIX 


SALT    SOLUTIONS 

Physiological  salt  solutions  for  immunity  work,  dilu- 
tion flasks,  etc.: 

Sodium  chloride,  c.p 8.5  gms. 

Distilled  water 1000.0  c.c. 

Chemically  pure  sodium  chloride  must  be  used  for 
immunity  work,  especially  for  animal  injection.  For 
dilution  flasks  the  best  grade  of  cooking  salt  serves  the 
purpose.  Salt  prepared  for  table  use  cannot  be  used  on  account 
of  its  starch  content. 

Normal  salt  solution  for  dilution  purposes,  etc., 
not  for  immunity  work: 

Sodium  chloride,  best  commercial  grade .       60  gms. 
Distilled  water 1000  c.c. 

Citrated  salt  solution  for  used  in  demonstrating  opso- 
nins: 

Sodium  chloride,  c.p 8.5  gms. 

Sodium  citrate 15.0  gms. 

Distilled  water 1000.0  c.c. 

TEST  SOLUTIONS 

Ehrlich's  test  solution  for  indol  production: 
Solution  I. 

Para-dimethyl-amido-benzaldehyde 4  gms. 

96%  alcohol 380  c.c. 

HC1,  cone..  .  . ....' 80  c.c. 

Solution  II.  Saturated  watery  solution  of  potassium 
persulphate  (oxidizing  agent). 

See  Exercise  44,  Part  I,  for  the  method  of  the  test. 


APPENDIX  371 

Nitrate  test  solutions: 
I.  Phenolsulphonic  acid. 

1.  Mix  3  gms.  of  pure  crystallized  phenol  with  37  gms. 
of  c.p.  concentrated  sulphuric  acid  (20.1  c.c.,  sp.gr.  1.84) 
in  a  round-bottom  flask. 

2.  Heat  for  six  hours  in  a  water  bath  at  100°  C.,  keeping 
the  flask  submerged  the  whole  time. 

This  may  crystallize  on  cooling,  but  it  can  be  brought 
into  solution  easily  by  heat. 

Directions  for  making  this  test  will  be  noted  in  Exercise 
45,  Part  I. 

II.  Diphenylamin.  A  solution  of  2%  diphenylamin  in 
sulphuric  acid  when  added  to  a  liquid  containing  nitrates 
or  nitrites  gives  a  blue  color. 

Diphenylamin 2  gms. 

Sulphuric  acid,  c.p.  cone 100  c.c. 

Nitrite  test  solutions: 

Solution  I.  8.0  gms.  sulphanilic  acid  dissolved  in  1000 
c.c.  of  5N  acetic  acid  (sp.gr.  1.041;. 

Solution  II.  5.0  gms.  a-naphthylamin  dissolved  in 
1000  c.c.  of  5N  acetic  acid.  These  solutions  should  be  kept 
separate  and  mixed  in  equal  parts  just  before  use. 

Nessler's  solution,  for  free  ammonia: 

1.  Dissolve  62.5  gms.  of  potassium  iodid  in  250  c.c.  of 
distilled  water.     Reserve  about  10  c.c.  of  this  solution. 

2.  Add  gradually  to  the  main  portion  a  cold  saturated 
solution  of  mercuric  chloride,  stirring  constantly  and  in- 
creasing the  quantity  of  mercuric  chloride  until  a  bright, 
permanent  precipitate  is  formed. 

3.  Now  add  the  reserved  potassium  iodid  solution  and 
again  add  the  saturated  mercuric  chloride  solution,  cautiously 
and  with  constant  stirring  until  a  distinct  though  slight 
red  precipitate  remains. 

4.  Dissolve  150  gms.  of  caustic  potash  in  150  c.c.  dis- 


372  APPENDIX 

tilled  water,  allow  the  solution  to  cool  and  add  it  to  the 
above  solution. 

5.  Dilute  to  one  liter  with  distilled  water. 

6.  Allow  to  stand  for  one  week  and  decant  for  use. 

MOUNTING  MEDIA 

Canada  balsam  for  making  permanent  mounts  of  mi- 
croscopic preparations : 

Canada  balsam,  dry,  hard,  for  microscopic  use 4  parts 

Xylol 3  parts 

This  gives  a  mounting  medium  of  about  the  right  con- 
sistency. It  should  not  "  thread  "  when  a  drop  is  taken 
out  with  the  glass  rod.  Balsam  should  be  kept  in  a  bottle 
stoppered  with  a  glass  bell-stopper,  and  having  a  rim 
arranged  so  that  the  excess  of  balsam  taken  upon  the 
glass  rod  can  be  drained  off. 

Immersion  oil  for  oil  immersion  objectives. 

It  is  necessary  that  the  immersion  oil  have  practically 
the  same  index  of  refraction  as  glass  in  order  to  avoid 
dispersion  of  any  of  the  light  rays.  Cedar  wood  oil  having 
a  refractive  index  of  1.515  to  1.520  is  the  usual  medium 
interposed  between  the  specimen  and  the  oil  immersion 
objective  as  it  has  approximately  the  same  index  of  refrac- 
tion as  crown  glass,  1.518.  The  refractive  index  of  air  is 
1.000. 

Chinese  ink: 

Bum's  "  Pelikan  "  Chinese  ink 1  part. 

Distilled  water 7  parts. 

Tube,  using  8  to  10  c.c.  per  tube,  sterilize  in  the  autoclav 
and  allow  to  stand  two  or  three  weeks  without  disturbing, 
for  sedimentation  to  take  place.  It  is  to  be  used  without 
shaking  or  disturbing  any  more  than  necessary. 


APPENDIX  373 


STAINS 

Methylen  blue  for  differentiating  living  from  dead 
yeast  cells: 

Methylen  blue 0.1  gm. 

Distilled  water. .  . 1000.0  c.c. 

Aqueous-alcoholic  stains,  fuchsin,  methylen  blue  and 
gentian  violet: 

1.  A  saturated  alcoholic  solution  of  a  stain  is  prepared 
by  shaking    frequently  about    10  gms.  of  the    stain  with 
100  c.c.  of  absolute  alcohol.     If  the  stain  dissolves  quickly, 
add  more  dry  stain.     The  alcoholic  solution    should    be 
slightly  supersaturated. 

2.  Allow  the  undissolved  stain  to  settle  over  night. 

3.  Decant. 

4.  Dilute  1  part  of  the  alcoholic  solution  with  9  parts 
of  distilled  water. 

Note  1.  If  95%  alcohol  is  used  instead  of  absolute  alcohol  to 
dissolve  the  stain,  the  dilution  should  be  made  1  :  7. 

Note  2.  These  aqueous  solutions  may  not  keep  longer  than  about 
a  month,  while  the  saturated  alcoholic  solutions  keep  indefinitely. 

Note  3.  The  vegetative  forms  of  bacteria  stain  more  or  less 
readily  with  all  aqueous-alcoholic  stains  but  not  with  saturated  alcoholic 
stains.  Acid-fast  bacteria,  e.g.,  Bact.  tuberculosis,  are  the  exception 
to  the  former. 

Anilin- water  gentian  violet: 

1.  Shake  5  c.c.  of  anilin  oil  vigorously  with  100  c.c.  of 
distilled  water  in  a  stoppered  bottle  for  several  minutes. 

2.  Filter  through  a  wet  filter  immediately  before  use. 

3.  Add  1  part  of  saturated  alcoholic  solution  of  gentian 
violet  to  9  parts  of  the  freshly  prepared  anilin-water  and 
filter  immediately  before  use. 

Note.  Anilin-water  stains  do  not  keep  longer  than  about  a  week. 
The  stock  solutions  will  keep  indefinitely  if  kept  separate. 


374  APPENDIX 

Ziehl-Nielson's  carbol-fuchsin. 

Solution  A. 

Basic  fuchsin 1  gm. 

Absolute  alcohol 10  c.c. 

Solution  B. 

Carbolic  acid 5  gms. 

Distilled  water 100  c.c. 

1.  Dissolve  the  fuchsin  in  the  absolute  alcohol.     (Solu- 
tion A.) 

2.  Dissolve   the    carbolic    acid   in   the    distilled   water 
(Solution  B). 

Note.    Solutions   A  and  B   will  keep   indefinitely  if  kept   sepa- 
rate. 

3.  Mix  in  the  proportion  of  10  c.c.  of  solution  A  to  100 
c.c.  of  solution  B. 

Note.     If  A  and  B  do  not  mix  readily,  warm  slightly  and  add  a 
few  drops  of  absolute  alcohol. 

4.  Filter. 

Loeffler's  alkaline  methylen  blue. 

Saturated  alcoholic  solution  of  methylen  blue 30  c.c. 

Potassium  hydrate,  0.1%  aqueous  solution 100  c.c. 


APPENDIX  375 


SOLUTIONS  FOR  USE  IN  STAINING 

Aceton-alcohol  for  decolorizing  in  Gram's  method  of 
staining: 

Aceton 10  c.c. 

Absolute  alcohol 100  c.c. 

Acetic  acid-alcohol  for  clearing  in  making  impression 
preparations  (also  used  for  decolorizing  in  ordinary  method 
of  spore-staining) : 

Alcohol,  90% 2  parts 

Acetic  acid,  1% 1  part. 

Mordant  for  staining  flagella: 

Tannin,  20% 10  c.c. 

Ferrous  sulphate,  cold  saturated  solution . .  8  c.c. 
Fuchsin,  cold  saturated  solution  in  absolute 

alcohol 1  c.c. 

Lugol's  iodin  solution,  for  use  in  Gram's  staining  method: 

lodin 1  gm. 

Potassium  iodid 3  gms. 

Distilled  water .  .  300  c.c. 


376 


APPENDIX 


STEAM  TEMPERATURE  PRESSURE  TABLE 


Temperature 
Centigrade. 

Mm.  of  Hg. 

Pounds  per  sq.in. 
Absolute  Pressure. 

Atmospheres. 

Degrees, 

98 

707.1 

13.7 

0.93 

99 

733.1 

14.2 

0.96 

100 

760.0 

14.7 

1.00 

101 

787.8 

15.2 

1.03 

102 

816.0 

15.8 

1.07 

103 

845.2 

16.3 

1.11 

104 

875.4 

16.9 

1.15 

105 

906.4 

17.5 

1.19 

106 

938.3 

18.1 

.23 

107 

971.1 

18.8 

.27 

108 

1004.9 

19.4 

.32 

109 

1039.6 

20.1 

.36 

110 

1075.3 

20.8 

.41 

111 

1112.0 

21.5 

.46 

112 

1149.8 

22.2 

.51 

113 

1188.6 

22.9 

.56 

114 

1228.4 

23.7 

.61 

115 

1269.4 

24.5 

.67 

116 

1311.4 

25.3 

.72 

117    , 

1354.6 

26.2 

.78 

118 

1399.0 

27.0 

.84 

119 

1444.5 

27.9 

.90 

120 

1491.2 

28.8 

.96 

121 

1539.2 

29.7 

2.02 

122 

1588.4 

30.7 

2.09 

123 

1638.9 

31.7 

2.15 

124 

1690.7 

32.7 

2.22 

125 

1743.8 

33.7 

2.29 

APPENDIX 


377 


FORMULAE  FOR  CONVERSION  OF  DEGREES  OF  TEMPER- 
ATURE ON  ONE  SCALE  INTO  DEGREES  ON  ANOTHER 

Centigrade  (Celsius)  scale:    Freezing-point  =  0°;  boiling-point  =  100°. 
Fahrenheit  scale :  Freezing-point  =  32  ° ;  boiling-point  =  212°. 

Reaumur:  Freezing-point  =  0°;  boiling-point  =  80°. 

(F-32)4 


Degrees  C  X 1 .8 +32  =  Degrees  F. 
F-32 


Degrees 


1.8 
RX9 


=  Degrees  C. 


+32  =  Degrees  F. 


Degrees 


9 
RX5 

4 
CX4 

5 


=  Degrees  R. 


Degrees  C. 


Degrees  R. 


ALCOHOL  BY  VOLUME 
TRALLES 

(From  the  Chemlker  Kalender.'publlshed  by  Julius  Springer,  Berlin.) 


Per 
Cent 
by  Vol. 

Specific 
Gravity. 

Per 
Cent 
by  Vol. 

Specific 
Gravity. 

Per 
Cent 
by  Vol. 

Specific 
Gravity. 

Per 

Cent 
by  Vol. 

Specific 
Gravity. 

1 

0.9976 

26 

0.9689 

51 

0.9315 

76 

0.8739 

2 

0.9961 

27 

0.9679 

52 

0.9295 

77 

0.8712 

3 

0.9947 

28 

0.9668 

53 

0.9255 

78 

0.8685 

4 

0.9933 

29 

0.9657 

54 

0.9254 

79 

0.8658 

5 

0.9919 

30 

0.9646 

55 

0.9234 

80 

0.8631 

6 

0.9906 

31 

0.9634 

56 

0.9213 

81 

0.8603 

7 

0.9893 

32 

0.9622 

57 

0.9192 

82 

0.8575 

8 

0.9881 

33 

0.9609 

58 

0.9170 

83 

0.8547 

9 

0.9869 

34 

0.9596 

59 

0.9148 

84 

0.8518 

10 

0.9857 

35 

0.9583 

60 

0.9126 

85 

0.8488 

11 

0.9845 

36 

0.9570 

61 

0.9104 

86 

0.8458 

12 

0.9834 

37 

0.9559 

62 

0.9082 

87 

0.8428 

13 

0.9823 

38 

0.9541 

63 

0.9059 

88 

0.8397 

14 

0.9812 

39 

0.9526 

64 

0.9036 

89 

0.8365 

15 

0.9802 

40 

0.9510 

65 

0.9013 

90 

0.8332 

16 

0  9791 

41 

0.9494 

66 

0.8989 

91 

0.8299 

17 

0.9781 

42 

0.9478 

67 

0.8965 

92 

0.8265 

18 

0.9771 

43 

0.9461 

68 

0.8941 

93 

0.8230 

19 

0,9761 

44 

0.9444 

69 

0.8917 

94 

0.8194 

20 

0.9751 

45 

0.9427 

70 

0.8892 

95 

0.8157 

21 

0.9741 

46 

0.9409 

71 

0.8867 

96 

0.8118 

22 

0.9731 

47 

0.9391 

72 

0.8842 

97 

0.8077 

23 

0.9720 

48 

0.9373 

73 

0.8817 

98 

0.8034 

24 

0.9710 

49 

0.9354 

74 

0.8791 

99 

0.7988 

25 

0.9700 

50 

0.9335 

75 

0.8765 

100 

0.7939 

378 


APPENDIX 


DEGREE  f~\  DEGREES 
-110 


220 - 


210 
200 
190 
180 

iro 

160 
150 
140 
13JO 
120 
110^ 


90- 
80 -g 
70  -= 
60 -g 
50  j 
40  - 


30-= 
20 -J 

10  -| 

F 


100 


70 


50 


30 


20 


10 


10 


C 


FIG.  74. — Comparison  Fahrenheit-Centigrade  Scale. 


APPENDIX  379 

METRIC  SYSTEM 
Linear  Measure 

1000  millimicrons  =  1  micron  (micromillimeter). 
1000  microns         =  1  millimeter. 

10  millimeters   =  1  centimeter. 

10  centimeters  =  1  decimeter. 

10  decimeters    =  1  meter. 

10  meters  =  1  decameter. 

10  decameters   =  1  hectometer. 

10  hectometers  =  1  kilometer. 

10  kilometers    =  1  myriameter. 

The  unit  of  length,  one  meter,  is  equal  to     QQQQQQ  Part  of  the 

distance  measured  on  a  meridian  of  the  earth  from  the  equator  to  the 
pole  and  equals  about  39.37  inches. 


1,000,000  sq.  millimicrons  =  1  sq.  . 

1,000,000  sq.  microns         =  1  sq.  millimeter. 
= 


Square  Measure 

micron. 

.  .  millime. 

100  sq.  millimeters   =  1  sq.  centimeter. 
100  sq.  centimeters  =  1  sq.  decimeter. 
100  sq.  decimeters    =  1  sq.  meter          =  1  centare 
100  sq.  meters  =  1  sq.  decameter  =  1  are. 

100  sq.  decameters   =  1  sq.  hectometer  =  1  hectare. 
100  sq.  hectometers  —  1  sq.  kilometer. 
100  sq.  kilometers     =  1  sq.  myriameter, 

Cubic  Measure 

1000  cubic  millimeters  =  1  cubic  centimeter. 
1000  cubic  centimeters  =  1  liter. 

10  liters  =  1  decaliter. 

100  liters  =  1  hectoliter. 

1000  liters  =  1  kiloliter  =  1  cu.  meter  =  1,000,000  c.c. 

The  unit  of  capacity  is  the  liter  and  represents  the  volume  of  a 
kilogram  of  water  at  its  maximum  density,  4°  C.  and  760  mm.  mercury 
pressure. 


380  APPENDIX 

METRIC  SYSTEM— Continued 
Weight 

The  unit  of  weight  is  the  gram  and  represents  the  weight  of  one 
cubic  centimeter  of  water  at  its  maximum  density,  4°  C.  and  760  mm. 
mercury  pressure. 

10  milligrams    =  1  centigram. 

10  centigrams  =  1  decigram. 

10  decigrams     =1  gram. 

10  grams  =  1  decagram. 

10  decagrams    =1  hectogram. 

10  hectograms  =  1  kilogram  =  1000  grams. 

10  kilograms     =  1  myriagram. 

10  myriagrams  =  1  quintal. 

10  quintals        =  1  millier  or  tonneau. 


LIST  OF   TEXT   AND   REFERENCE   BOOKS 

ABBOT,  A.  C.     The  Principles  of  Bacteriology.     9th  Ed.     1915. 
AIRMAN,  C.  M.     Milk,  its  Nature  and  Composition.     1909. 
American  Public  Health  Association.     Standard  Methods  for  the 
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BASTIAN,  H.  C.     The  Beginnings  of  Life.     1872. 

BAYLISS,  W.  M.    The  Nature  of  Enzyme  Action.     3d  Ed.     1914. 

BAYLISS,  W.  M.     Principles  of  General  Physiology.     1915. 

BELCHER,  S.  D.     Clean  Milk.     1912. 

BESSON,  A.  Practical  Bacteriology,  Microbiology  and  Serum 
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BORDET,  J.  and  GAY,  F.  P.     Studies  in  Immunity.     1st  Ed.    1909. 

BOSTON,  L.  NAPOLEON.     Clinical  Diagnosis.     1905. 

BOWHILL,  T.     Manual  of  Bacteriological  Technique.  2d  Ed.   1902. 

BRACHVOGEL,  JOHN  K.  Industrial  Alcohol:  Its  Manufacture  and 
Uses.  1907. 

BRANNT,  WM.  T.  Vinegar,  Acetates,  Cider,  Fruit  Wines,  Pres- 
ervation of  Fruits.  2d  Ed.  1900. 

BUCHANAN,  ESTELLE  D.  and  R.  E.  Household  Bacteriology.    1913. 

BUCHANAN,  R.  E.    Veterinary  Bacteriology.     1916. 

BURGESS,  P.  S.     Soil  Bacteriology  Laboratory  Manual.     1914. 

CALKINS,  GARY  N.    Protozoology.     1909. 

CHAPIN,  CHARLES  V.     The  Sources  and  Modes  of  Infection.    2d 

Ed.     1912. 
Chemical  Rubber  Co.,  Cleveland,  Ohio.    Handbook  of  Chemistry 

and  Physics.     1914. 

CHESTER,  F.  D.    A  Manual  of  Determinative  Bacteriology.     1901. 
CITRON,  JULIUS.     Immunity.    Translated  by  A.  L.  Garbat.     1912. 
COHNHEIM,  OTTO.     Enzymes.     1st  Ed.     1912. 
CONN,  H.  W.     Agricultural  Bacteriology.     2d  Ed.     1909. 
CONN,  H.  W.    Bacteria  in  Milk  and  its  Products.     1903. 
CONN,  H.  W.    Bacteria,  Yeasts  and  Molds.     1903. 

381 


382         LIST  OF  TEXT  AND  REFERENCE  BOOKS 

CONN,  H.  W.   Bacteria,  Yeasts,  and  Molds.     1913. 

CONN,  H.  W.     Practical  Dairy  Bacteriology.     1910. 

CONN,  H.  W.,  ESTEN,  W.  M.,  and  STOCKING,  W.  A.  Classification 
of  Dairy  Bacteria.  1906.  Reprint  from  the  Report  of  the 
Storrs  (Connecticut)  Agr'l  Exp't,  Sta.  for  1906, 

DE  BARY,  H.     Comoarative    Morphology    and    Biology    of    the 

Fungi,  Mycetozoa  and  Bacteria.     1887. 
DE  BARY,  H.     Lectures  on  Bacteria.    2d  Ed.     1887. 
DECKER,  JOHN  W.    Cheese-making.     1905. 
DIEUDONNE,  H.    Bacterial  Food  Poisoning.     1909. 
DOANE,  R.  W.    Insects  and  Disease.     1910. 
DON,  JOHN,  and  CHISHOLM,  JOHN.    Modern  Methods  of  Water 

Purification.     1911. 
DUCKWALL,  E.  W.     Canning  and  Preserving  of  Food  Products 

with  Bacteriological  Technique.     1905. 
DUGGAR,  B.  M.     Fungous  Diseases  of  Plants.     1909. 
DUGGAR,  B  M.    Plant  Physiology.     1912. 
DtiRCK,  HERMANN.    Allgemeine    Pathologische    Histologie.     Teil 

III.     1903. 

EFFRONT,  JEAN.  Enzymes  and  Their  Applications.  1st  Ed. 
1904. 

EHRLICH,  P.  and  BOLDUAN,  CHARLES.  Collected  Studies  in  Im- 
munity. 2d  Ed.  1910. 

ELLIOTT,  S.  MARIA.  Household  Bacteriology.  1914.  From 
Handbook  of  Health  and  Nursing.  Edited  by  the  American 
School  of  Home  Economics.  1912. 

ERNST,  WILLIAM.  Milk  Hygiene.  Transl.  by  John  R.  Mohler 
and  Adolph  Eichhorn.  1914. 

EULER,  HANS.  General  Chemistry  of  the  Enzymes.  Translated 
by  Thomas  H.  Pope.  1st  Ed.  1912. 

EYRE,  J,  W,  H.    Bacteriological  Technique.    2d  Ed.  1913. 

FARRINGTON,  E.  H.,  and  WOLL,  F.  W.    Testing  Milk  and  its 

Products.     1904. 
FISCHER,  ALFRED.     Structure  and  Functions  of  Bacteria.     Transl. 

by  A.  Coppen  Jones.     1900. 
FLEISCHMANN,  W.    The  Book  of  the  Dairy,  1896.    Transl.  by  C. 

M.  Aikmann  and  R.  Patrick  Wright. 


LIST  OF  TEXT  AND  REFERENCE  BOOKS         383 

FLUGGE,  C.    Die  Mikroorganismen,  1896. 
FOWLER,  G.  J.    Bacteriological  and  Enzyme  Chemistry.     1911. 
FRED,  E.  B.     Laboratory  Manual  of  Soil  Bacteriology.     1916. 
FROST,  W.  D.    A  Laboratory  Guide  in  Elementary  Bacteriology. 

3d  Ed.     1904. 
FROST,  W.  D.  and  MCCAMPBELL,  E.  F.    A  Text-book  of  General 

Bacteriology.     1910. 

FUHRMANN,  FKANZ,    Vorlesungen  uber  bakterien  Enzyme,    1907. 
FUHRMANN,     FRANZ.    Vorlesungen  uber   technische    Mykologie. 

1913. 
FULLER,  G.  W.     Sewage  Disposal.     1912. 

GAGE,  S.  H.    The  Microscope1.     1904. 

GERHARD,  WM.  P.    The  Sanitation,    Water   Supply   and   Sewage 

Disposal  of  Country  Houses.     1909. 

GORHAM,  F.  P.     Laboratory  Course  in  Bacteriology.     1915. 
GREEN,    J.    REYNOLDS.    Soluble    Ferments    and    Fermentation. 

2d  Ed.     1901. 
GUEGUEN,  F.    Les   Champignons:    Parasites    de    1'Homme    and 

des  Animaux.     1904. 
GUILLIERMOND,  A.    Les  Levures.     1912. 

HANSEN,  EMIL  CHR.    Practical  Studies  in  Fermentation.    1896. 
HARDEN,  ARTHUR.    Alcoholic  Fermentation.     2d  Ed.     1914. 
HASTINGS,  E.  G.  and  WRIGHT,  W.  H.    A  Laboratory  Manual  of 

General  Agricultural  Bacteriology.     1913. 
HAWK,  P.  B.     Physiological  Chemistry.    4th  Ed.     1913. 
HEINEMANN,  P.  G.    A  Laboratory  Guide  in  Bacteriology.    2d  Ed. 

1911. 

HERTWIG,  OSCAR.    Allgememe  Biologic.     1912. 
HERZOG,  M.     Disease-Producing  Microorganisms.     1910. 
HEWLETT,  R.  T.     Manual  of  Bacteriology,  5th  Ed.     1915. 
Hiss,  P.  H.  and  ZINSSER,  H.    Text-book  of  Bacteriology.     2d  Ed. 

1914. 

HOOKER,  A.  H.     Chloride  of  Lime  in  Sanitation.     1913. 
HUEPPE,  F.     Methods  of  Bacteriological  Investigation.    Transl. 

by  H.  M.  Biggs.     1886. 

IAGO,  WM.  and  IAGO,  WM.  C.    The  Technology  of  Bread-making. 
1911. 


384         LIST  OF  TEXT  AND  REFERENCE  BOOKS 

JENSEN,  C.  0.    Essentials  of  Milk  Hygiene.     Transl.  by  Leonard 

Pearson.     1907. 
JESS,    PAUL.     Kompendium   der   Bakteriologie   und   Blutserum- 

therapie.     1903. 

JORDAN,  EDWIN  0.     General  Bacteriology.     3d  Ed.     1914. 
JORGENSEN,  A.     Microorganisms  and  Fermentation.     Transl.  by 

A.  K.  Miller  and  E.  A.  Lennholm.     1893. 

KERSHAW,  G.  B.    Modern  Methods  of  Sewage  Purification.     1911. 
KINNICUTT,  L.  P.,  WINSLOW,  C.  E.  A.,  and  PRATT,  R.  W.    Sewage 

Disposal.     1910. 
KISSKALT,  K.,  und  HARTMANN,  M.    Praktikum  der  Bakteriologie 

und  Protozoologie.     1907. 
KITT,    TH.     Bakterienkunde    und     pathologische    Mikroskopie. 

1903. 

KITT,  TH.    Text-book  of  Comparative  General  Pathology.     Trans- 
lated by  Wm.  W.  Cadbury  and  Allen  J.  Smith.     1906. 
KLOCKER,    ALB.     Fermentation    Organisms.     Translated    by    G. 

E.  Allen  and  J.  H.  Millar.     1904. 
KOLLE,  W.,   und  WASSERMANN,  A.    Handbuch  der  pathogenen 

Microorganismen  I,  II  und  III  und  Atlas.     1903. 
KOLMER,  J.  A.     Infection,  Immunity  and  Specific  Therapy.    1915. 
KRAUS,  R.,  und  LEVADITI,  C.    Handbuch  der  Technik  und  Metho- 

dik  der  Immunitatsforschung.    Bd.  I,  Antigene.    1908;    Bd. 

II,  Antikorper.     1909;    Erster  Erganzungsband.     1911. 
KRUSE,  W.    Allgemeine  Mikrobiologie.     1910. 

LAFAR,  FRANZ,    Die  Essigsaure-Garung.     1913. 

LAFAR,    FRANZ.     Technische    Mykologie.     German  Ed.     Bd.    I, 

1904-1907;     Bd.    II,  1905-1908;    Bd.  Ill,  1904-1906;    Bd. 

IV,  1905-1907;    Bd.    V,    1905-1914.     English  Ed.    Vol.  I, 

1898;  Vol.  II,  Part  1, 1903;  Vol.  II,  Part  II,  1910.    Translated 

by  Charles  T.  C.  Salter. 
LAW,    JAMES.    Veterinary    Medicine.     3d    Ed.    Vol.    I.     1910; 

Vol.  II,  1911. 
LEE,   H.   BOLLES.    The  Microtomist's  Vade  Mecum.    6th  Ed. 

1905. 
LEFAS,  E.     La  Technique  histo-bacteriologique  moderne.     1906. 


LIST  OF  TEXT  AND  REFERENCE  BOOKS          385 

LEHMANN,  K.  B.,  u.  NEUMANN,  R.  O.  Bakteriologie  und  bak- 
teriologische  Diagnostik.  Teil  I,  Atlas,  1910;  Teil  II,  Text, 
1912. 

LIPMAN,  J.  G.    Bacteria  in  Relation  to  Country  Life.     1908. 

LIPMAN,  J.  G.,  and  BROWN,  P.  E.  Laboratory  Guide  in  Soil  Bacter- 
iology. 1911. 

LOEB,  JACQUES.    The  Mechanistic  Conception  of  Life.     1912. 

LOEB,  JACQUES.     Studies  in  General  Physiology,  Part  I.     1905. 

LOHNIS,  F.  Handbuch  der  landwirtschaftlichen  Bakteriologie. 
1910. 

LOHNIS,  F.  Laboratory  Methods  in  Agricultural  Bacteriology. 
Translated  by  Wm.  Stevenson  and  J.  Hunter  Smith.  1913. 

LOHNIS,  F.  Vorlesungen  iiber  landwirtschaftliche  Bakteriologie. 
1913. 

MACFADYEAN,  ALLAN.     The  Cell  as  the  Unit  of  Life.     1908. 

MARSHALL,  C.  E.     Microbiology.     1911. 

MASSEE,  G.     Diseases  of  Cultivated  Plants  and  Trees.     1914. 

MAST,  S.  0.     Light  and  the  Behavior  of  Organisms.     1911. 

MATTHEWS,  C.  G.     Manual  of  Alcoholic  Fermentation.     1901. 

McCAMPBELL,  E.  F.  Laboratory  Methods  for  the  Experimental 
Study  of  Immunity.  1909. 

McFARLAND,  JOSEPH.  Pathogenic  Bacteria  and  Protozoa.  7th 
Ed.  1912. 

MCFARLAND,  JOSEPH.  Biology,  General  and  Medical.  2d  Ed. 
1914. 

McKAY,  G.  L.,  and  LARSEN,  C.  Principles  and  Practice  of  Butter- 
making,  1913. 

MERCK'S  INDEX.     1907. 

METCHNIKOFF,  ELIE.  Comparative  Pathology  of  Inflammation. 
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METCHNIKOFF,  ELIE.     Immunity  in  Infective  Diseases.     1905. 

MIGULA,  W.    System  der  Bakterien.    Bd.  I,  1897;   Bd.  II,  1900. 

MOLISCH,  HANS.    Die  Eisenbakterien.     1910. 

MOLISCH,  HANS.    Leuchtende  Pflanzen.     1904. 

MOLISCH,  HANS.     Die  Purpurbakterien.     1907. 

MOORE,  V.  A.  The  Pathology  and  Differential  Diagnosis  of 
Infectious  Diseases  of  Animals.  1916. 

MOORE,  V.  A.    Principles  of  Microbiology.     1912. 


386          LIST  OF  TEXT  AND   REFERENCE  BOOKS 

MOORE,  V.  A.    Bovine  Tuberculosis  and  its  Control.     1913. 
MOORE,  V.  A.  and  Fitch,  C.  P.    Bacteriology  and  Diagnosis. 

1914. 
Mum,  ROBERT,  and  RITCHIE,  JAMES.    Manual  of  Bacteriology. 

6th  Ed.     1913. 
MULLER,  PAUL  TH.    Vorlesungen  iiber  Infektion  und  Immunitat. 

1904. 

NOCARD,  Ep.,  and  LECLAINGHE,  E.    Les  Maladies  imcrobiennes 

des  Animaux.     T.  I,  1903;    T.  II,  1903. 
NOVY,  F.  G.    Laboratory  Work  in  Bacteriology.     1899. 
NUTTALL,  G.  H.  F.     Blood  Immunity  and  Blood  Relationship: 

Precipitin  Tests.     1904. 

OGDEN,   HENRY   N.   and   CLEVELAND,    H.   BURDETT.    Practical 

Methods  of  Sewage  Disposal.  1912. 
OPPENHEIMER,  C.     Die  Fermente  und  ihre  Wirkungen.     Bd.  I, 

Vierte  Auflage.     1913;  Bd.  II,  1913. 

PARK,  WM.  H.  Pathogenic  Bacteria  and  Protozoa.  4th  Ed. 
1910. 

PASTEUR,  L.  Studies  on  Fermentation.  Translated  by  F.  Faulk- 
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PFEIFFER,  L.     Die  Protozoen  als  Krankheitserreger.     1890. 

PORCHER,  CH.    Le  Lait  Desseche.     1912. 

PRESCOTT,  S.  C.  and  WINSLOW,  C.-E.  A.  Elements  of  Water 
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RICHMOND,  H,  D,    Dairy  Chemistry,    1899. 

RICKETTS,  H.  T.  and  DICK,  G.  F.    Infection,  Immunity  and  Serum 

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RIDEAL,    SAMUEL.    Sewage    and    the    Bacterial    Purification    of 

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ROGERS,  A.,  and  AUBERT,  A.  B.    Industrial  Chemistry.     1912. 
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LIST  OF  TEXT  AND  REFERENCE  BOOKS          387 

ROSENAU,  M.  J.     Preventive  Medicine  and  Hygiene.     1913. 

RUSSELL,  H.  L.,  and  HASTINGS,  E,  G.  Experimental  Dairy  Bac- 
teriology. 1909. 

RUSSELL,  H.  L.,  and  HASTINGS,  E.  G.  Outlines  of  Dairy  Bacter- 
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RUSSELL,  H.  L.,  and  HASTINGS,  E.  G.  Agricultural  Bacteriology. 
1915. 

SADTLER,  S.  P.    Industrial  Organic  Chemistry.     1912. 

SANTEE,  E.  M.    Farm  Sewage.     1912. 

SAVAGE,   WM.    G.    The   Bacteriological   Examination   of   Water 

Supplies.     1906. 
SAVAGE,  WM.  G.    The  Bacteriological  Examination  of  Food  and 

Water.     1914. 

SAVAGE,  WM.  G.     Milk  and  the  Public  Health.     1912. 
SCHNEIDER,  ALBERT.     Bacteriological  Methods  in  Food  and  Drug 

Laboratories.     1915. 

SHINKLE,  C.  A.  American  Commercial  Methods  of  Manufactur- 
ing Preserves,  Pickles,  Canned  Foods,  etc.  Revised  Ed. 

1912. 
SMITH,  ERWIN  F.    Bacteria  in  Relation  to  Plant  Diseases.    Vols. 

I,  1905;   II,  1911;   III,  1914. 
SNYDER,  HARRY.     Dairy  Chemistry.     1911. 
STERNBERG,  G.  M.    Textbook  of  Bacteriology.     1896. 
STEVENS,  F.  L.,  and  HALL,  J.  G.    Diseases  of  Economic  Plants. 

1913. 
STITT,  E.  R.    Practical  Bacteriology,  Blood  Work  and  Animal 

Parasitology.    3d  Ed.     1914. 
SYKES,  W.  J.    The  Principles  and  Practice  of  Brewing.     1897. 

THORP,  F.  H.     Outlines  of  Industrial  Chemistry.     1905. 
THRESH,  JOHN  C.     Examination  of  Waters  and  Water  Supplies. 

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TYNDALL,   JOHN.     Floating   Matter   of  the  Air  in   Relation   to 

Putrefaction  and  Infection.     1881. 

VAN  SLYKE,  Lucius  L.,  and  PUBLOW,  CHARLES  A.    The  Science 

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388         LIST   OF  TEXT  AND  REFERENCE  BOOKS 

VAUGHAN,  V.  C.,  VAUGHAN,  V.  C.,  Jr.,  and  VAUGHAN,  J.  W.  Pro- 
tein Split  Products  in  Relation  to  Immunity  and  Disease. 
1913. 

VERNON,  H.  M.     Intracellular  Enzymes.     1908. 

WALKER,  E.  W.  AINLEY.    Inflammation,  Infection  and  Fever. 

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WARBASSE,  J.  P.    The  Conquest  of  Diseases  Through  Animal 

Experimentation.     1910. 

WARD,  A.  R.    Pure  Milk  and  the  Public  Health.     1909. 
WARD,  H.  MARSHALL.     Diseases  in  Plants.     1901. 
WASSERMAN,  A.     Immune  Sera.    Translated  byChas.   Baldwin. 

1904. 

WHIPPLE,  G.  C.     Typhoid  Fever.     1st  Ed.     1908. 
WILLIAMS,    H.    U.     Manual    of    Bacteriology.     Revised    by    B. 

Meade  Bolton.     5th  Ed.     1908. 

WILLOUGHBY,  EDWARD  F.     Milk:  Its  Production  and  Uses.     1903. 
WILSON,  EDMUND  B.    The  Cell  in  Development  and  Inheritance. 

1911. 

WING,  HENRY  H.    Milk  and  its  Products.     1904. 
WINSLOW,  C.  E.  A.,  and  WINSLOW,  ANNE  R.    The  Systematic 

Relationships  of  the  Coccacese.     1908. 

ZIEGLER,    ERNST.    Pathologic    und     Anatomie.     Bd.     I,    1901; 

Bd.  II,  1902. 

ZINSSER/HANS.     Infection  and  Resistance.      1915. 
ZOPF,  WILHELM,    Die  Pilze.     1890. 

PERIODICALS 

Annales  de  1'Institut  Pasteur. 

Arbeiten  aus  dem  kaiserlichen  Gesundheitsamte. 

Archiv  fur  experimentale  Pathologie  und  Pharmakologie. 

Archiv  fur  Hygiene. 

Archiv  fiir  Schiffs  und  Tropenhygiene. 

Berliner  klinische  Wochenschrift. 
Biochemische  Zeitschrift. 
British  Medical  Journal. 
Bulletin  de  1'Institut  Pasteur. 


LIST  OF  TEXT  AND  REFERENCE  BOOKS         389 

Centralblatt  fur  Bakteriologie,  I  Abteilung,  Originate.  Medizin- 
isch-hygienische  Bakteriologie  und  tierische  Parasitenkunde. 

Centralblatt  fur  Bakteriologie,  I  Abteilung,  Referate. 

Centralblatt  fur  Bakteriologie,  II  Abteilung.  Allgemeine  land- 
wirtschaftlich-technologische  Bakteriologie,  Garungs-phy- 
siologie  und  Pflanzenpathologie. 

Comptes  Rendus  Academic  des  Sciences. 

Comptes  Rendus  Societie  de  Biologic. 

Comptes  Rendus  des  Travaux  du  Laboratoire  de  Carlsberg. 

Deutsche  medizinische  Wochenschrift. 
Experiment  Station  Record. 
Hygienische  Rundschau. 

Jahresbericht  liber  die  Forsch.  d.  path.  Mikroorganismen,  Baum- 

garten's. 
Jahresbericht  tiber  die  Fortschritte  der  Lehre  von  den  Garungs 

Organismen,  Koch's. 
Journal  of  Agricultural  Research. 
Journal  of  the  American  Medical  Association. 
Journal  of  the  American  Veterinary  Medical  Association. 
Journal  of  Experimental  Medicine. 
Journal  of  Hygiene. 
Journal  of  Infectious  Diseases. 
Journal  of  Medical  Research. 
Journal  of  Pathology  and  Bacteriology. 
Journal  of  the  Royal  Army  Medical  Corps. 
Journal  of  Tropical  Medicine. 

Lancet. 

Proceedings  of  the  Royal  Society  of  London. 

Revue  ge'ne'rale  du  Lait. 

Zeitschrift  fur  Hygiene  und  Infektionskrankheiten. 
Zeitschrift  fur  Immunitatsforschung. 


STRtrCTtTKE    OF   COLONIES. 

1.  Areolate. 

2.  Grumose. 

3.  Moruloid. 

4.  Clouded. 

5.  Gyrose. 

6.  Marmorate. 

7.  Reticulate. 


EDGES  OF  COLONIES. 

8.  Repand. 

9.  Lobate. 

10.  Efose. 

11.  Auriculate. 

12.  Lacerate. 

13.  Fimbriate.* 

14.  Ciliate.* 


*  For  illustration  see  next  page. 


APPENDIX 


391 


13 


14 


B 


ELEVATION  OF  COLONIES. 

1.  Flat. 

2.  Raised. 

3.  Convex. 

4.  Pulvinate. 

5.  Hemispherical. 

6.  Umbilicate. 

7.  UmbonatCk 

8.  Concave. 

9.  Rectangular  depression. 


392 


APPENDIX 


APPENDIX 


393 


INDEX 


Abortion,    contagious,    diagnosis 

of,  329 
Accidents,  X 
Acetacidase,  181,  183 
Acetaldehydase,  183 
Acetaldehyde,  183 
Acetic  acid-alcohol,  375 
Aceton-alcohol,  96,  375 
Acid,  acetic,  183,  215,  371 

acetic,  enzyme  of,  181 

acetic,  glacial,  97 

carbolic,  48,  185-188,  210-212, 
303,  308,  364,  365,  371,  374 

citric,  354 

digallic,  enzyme  of,  180 

fatty,  183 

hydrochloric,  39,  245,  313 

hydrochloric,  effect  on  pigment, 
175,  176 

hydrochloric,  N/l,  20,  367,  368 

hydrochloric,    N/20,    20,    245, 
367,  368 

hydrocyanic,  183 

lactic,  23,  181,  184,  218 

lactic,  enzyme  of,  181 

normal,  20-22,  367,  368 

oleic,  183 

Acidophile,  154,  184 
Acid,  oxalic,  313 

palmitic,  183 

phenolsulfonic,  133,  371 

picric,  87 

pyrogallic,  156,  160,  165,  248 


Acid,  silicic,  colloidal,  state  of,  33 

stearic,  183 

sulfanilic,  133,  251,  371 

sulfuric,  concentrated,  213,  249, 
308,  366,  371 

sulfuric,  for  decolorization,  92 
Acid-fast  bacteria,  92,  97,  324 

stain,  135 

Acid  production,  23, 112,  137, 169 
Acid-proteinase,  180 
Acidity,  20-22 

of  fresh  beef,  29 
Acids,  184 

a-amino,  180 

organic,     decomposition,     169, 
184 

organic,  enzymes  of,  180,  181 
Action,  selective,  170 
Activator,  193,  194 
Adhesion    culture,    78,    79,    100, 

107,   120 
Adjustment,  coarse,  65-69,  77 

fine,  65-69,  77 

of  reaction,  21,  22,  30 
Aeration  of  soils,  245,  246 
Aerobic  bacteria,  46,  154,  246,  247 
Aerogenic,  154 
Aeroscope,  sand  filter,  221 
Agar,  37-41 

action  of  acid  on,  38,  39 

as  a  microbial  food,  40,  41 

commercial  form  of,  38 

Congo  red,  355 

Conradi-Drigalski's,  361,  363 
395 


396 


INDEX 


Agar,  dextrose,  114,  175 

dextrose     calcium     carbonate, 

167,  349 

digestion  of,  39,  40 
Endo,  361,  363 
fermented,  41,  349 
filterability  of,  39,  40 
litmus  lactose,  349,  357,  358 
mannit,  253,  254 
melting  point  of,  39 
nitrate,  251,  354 
nitrogen-free  ash,  256,  355 
nutrient,  19,  20,  41-43 
nutrient,  preparation  of,  41-43 
percentage  used  in  media,  40 
soil  extract,  263,  351 
solidification  of,  39,  40 
solubility  of,  39,  40 
source  of,  37 
sterilization  of,  40 
urea,  352 
washing  of,  41 
waste,  IX 
plate   culture,   preparation   of, 

129 
shake,  litmus  lactose,  223,  228, 

236,  357 
slant,  26,  43 
Agents,  chemical,  of  sterilization, 

5,  14 

physical,  of  sterilization,  5 
sterilizing,  5 
Agglutination,   macroscopic  test, 

310-312 

microscopic  test,  312 
observation  of,  74 
Air,    bacterial  analysis    of,  220- 

222 

displacement  of,  157,  164,  165 
exclusion  of,  156,  157 
exhaustion  of,  156 
microorganisms    in,    146,    147, 

220-222,  272,  273 


Air,  relation  to  pigment  formation, 

175 
Albumin,  egg,  use  of,  29 

in  milk,  23 
Albumins,  coagulation  by  heat,  28 

soluble,  as  food,  172 
Albuminoids,  32 

effect  of,  on  sterilization,  8 
Alcohol,  by  volume,  table  of,  377 

ethyl,    181-183,    215-217,   364, 
369 

ethyl,  enzyme  of,  181,  183 

ethyl,  fermentation  of,  183 

for  cleaning  microscope,  65 

for  decolorization,  265,  266 

for  drying,  3,  4,  16 

for  extraction  of  fat,  92 

for  producing  vacuum,  156 

sterilization  in,  6,  14,  257 
Alcoholase,  181,  183 
Alcoholic  fermentation,  114,  122, 

171 

Alcoholoxidase,  181 
Aldehydases,  181,  183 
Aldehydes,  182 
Alkali,  action  of  on  methylen  blue, 

87 
Alkali,  free,  1 

normal,  20-22,  368 

production,  23 
Alkali-proteinase,  180 
Amidases,  181 
Amides,  acid,  181 
Amino  acids,  enzymes  of,  180 
Ammonia,  37,  44,  131,  134 

formation  in  soils,  245,  246,  252 
Ammonium  carbonate,  181,  185 

hydroxide,  134,  245,  246 

malate,  353 

nitrate,  246 

sulfate,  170,  308,  350,  352,  353 

tartrate,  350 
Amoebobacter,  348 


INDEX 


397 


Amphoteric  reaction  of  casein,  24 

Amygdalase,  180 

Amygdalin,  enzyme  of,  180,  182 

Amylase,  179,  182 

Anaerobe,  154 

obligate,  154 

partial,  154 
Anaerobic  bacteria,  46,  154,  248 

culture  methods,  155-167 
ANDERSON,  JOHN  F.  and  McCLiN- 

TIC,  THOS.  B.,  213      . 
Anesthesia,     general,    295,    296, 
299 

local,  295,  299 
Anesthetic,  295,  296,  309 

administration  of,  295,  296 
Anilin,  basis  of  dyes,  87 

dyes,  87 

oil,  373 
Anilin-water,  gentian  violet,  373 

preparation  of,  373 

stains,  72,  94,  373 
Animal,  fluids  as  media,  19 

inoculation,  295-301 

method  of  holding  for  inocula- 
tion, 296 

preparation     for     inoculation. 
295-301 

tissues,  as  media,  19,  20 
Animals,    autopsied,    destruction 
of,  6 

inoculated,  care  of,  301 
Anjesky's  spore-staining  method, 

90,303 

Anthrax,  302,  303 
Antibiosis,  45,  216 
Antibodies,  313-315,  328 
Antigen,    for   agglutination   test, 
311 

for   complement   fixation   test, 
325-327 

preservation  of,  326 

titration  of,  327 


Antiopsonic,  321 

Antiseptic,  184 

Antiseptics,  sterilization  by,  15 

Anti-serum,  310,  322 

Antitoxin,  309 

tetanus,  309,  310 
Apparatus,  sterilization  of,  6,  7 
Aqueous-alcoholic  stains,  72,  87, 

88 

Are,  379 

Arnold  sterilizer,  9,  10 
Arrangement  of  microorganisms, 

74 

Artery,  femoral,  298 
Asbestos,  shredded,  as  filter,  13 
Aseptic,  IX 
Ash,  in  milk,  23 
Asparagin,  173,  350,  356 
Aspergillus  niger,  -100,  101,   104, 

105,  107,  173,  199,  207 
Aspiration,  for  filtration,  13 
Autoclav,  IX,  11,  28 
Auxanography,  173 
Azolitmin,  source  of,  369 

solution,  Kahlbaum's,  -25,  181, 
369 

solution,  purpose  of,  369 
Azotobacter,  253-255,  354,  355 

chroococcum,  254 

B 

Bacillus,  meat,  197 

slimy  milk,  197 

Bacillus  acidi    lactiti,   347,    362, 
363 

aerogenes,  347,  362,  363 

aerogenes  2,  362,  363 
Bacillus  alvei,  97 

amylovorus,  97 

caratovorus,  97,  291-294 

cholerce  suis,  97,  310,  311,  362, 
363 

cloaca,  362,  363 


398 


INDEX 


Bacillus  coli  communior,  362,  363 
coli  communis,  47,  97,  145,  146, 

152,  153,  154,  174,  188,  191, 

195,  196,  223-233,  289,  290, 

347,  352,  359-363 
cyanogenus,  175 
erythrogenes,  352 
fluorescens  liquefaciens,  187,  347 
gelaticus,  n.  sp.  (gran),  41 
indicus,  155 

lactis  aerogenes,  362,  363 
megaterium,  97,  185,  199 
mesentericus  vulgatus,  97,  174 
mycoides,  63,  97,  174,  187,  191, 

200,  201,  245,  264,  347 
oligocarbophilus,  250 
paralyphosus  A,  362,  363 
paratyphosus  B,  362,  363 
pasteurii,  352,  353 
phytophthorus,  97 
prodigiosus,  155,  170,  175,  176, 

185,  187,  195,  196,  205 
proteus,  97,  352 
radiobacter,  254,  255 
ramosus,  187,  188 
ruber,  155 
subtilis,  63,  97,  164,  173,  187, 

197,  198,  205,  264 
tetani,  97,  308 
typhosus,  97,  200,  201,  208,  209, 

210,  211,  227,  228,231,  233, 

310,  311,  347,  359-363 
violaceus,  155,  175,  188,  197 
vulgaris,  97 
Bacteria,  acetic,  183 
acid-fast,  92 
aerobic,  46 
anaerobic,  46 
biochemical  activities  of,  23-25, 

182-185 

chromogenic,  242 
compared  with  protozoa,   140, 

141 


Bacteria,  effect  of  desiccation  on, 
197, 198 

effect  of  moist  and  dry  heat  on, 
202,  203 

flagella  of,  93 

Gram-negative,  95-97 

Gram-positive,  95-97 

green,  154 

identification  of,  125-138 

in  air,  146,  147,  220-222 

longevity  of,  197,  198 

nitrate,  249-251,  353 

nitrite,  249-251,  353 

pathogenic,  125,  221,  301 

pathogenic,    isolation   of,    301, 
302 

phosphorescent,  177,  178 

purple,  154 

slime-forming,  97 

soil,  241-244 

spores  of,  destruction  by  heat 
5-11 

study  of,  125-132,  135-138 

sulfur,  154,  348 

true,  347,  348 

urea,  185 

vegetative    forms,    destruction 
of,  by  heat,  5-11 

vinegar,  183,  184,  192,  193 

water,  identification  of,  361-363 

weight  of,  152,  153,  243,  244 
Bacleriacece,  347 
Bacterins,  autogenous,  316,  317 

polyvalent,  318 

preparation  of,  316-318 

preservation  of,  15 

stock,  317 

Bacteriopurpurin,  348 
Bacterium  abortus,  163,  325,  347 

aceti,  192,  215,  216 

acidi  lactici,  97 

aerogenes,  97,  198,  227,  228,  231, 
233,  362,  363 


INDEX 


399 


Bacterium  anthrads,  97,  98,  302, 

303 

bulgaricum,  96,  97,  347,  349 
lactis  acidi,   167-169,  184,  185, 
188,  191,  199,  214,  215,  217- 
219,  289,  347,  349 
mallei,  97,  327,  328 
nenckii,  41 
tuberculosis,  90,  92,  97,  303-305, 

347,  373 

tumefaciens,  258,  259,  355 
Balsam,  Canada,  65,  71,  372 
Banana,  as  a  medium,  20 
Base,  20 

Bath,  running  water,  36 
Beaded,  87 

Bedding,  sterilization  of,  10 
Beef,  chopped  lean,  28 
Beer  fermentation,  182 
manufacture,  yeasts  used,  114 
wort,  22 
Beggiatoa,  348 
BeggiatoacecB,  348 
BEIJERINCK,  41,  155,  170,  174 
Bengal  isinglass,  37 
BENIANS,  T.  H.  C.,  97 
Benzaldehyde,  182 
Benzol,  effect  on   pigment,  175, 

176 
Berkefeld  filter,  238,  303,  304 

filters,  sterilization  of,  6 
BESSON,  A.,  15,  19,  133,  140,  147, 
149,  151,  167,  222,  303,  305, 
308,  310 

Bile,  esculin,  360,  362,  363 
isolation    of    pathogens    from, 

302 

litmus  lactose,  223,  360 
salts,  value  in  media,  360 
Biochemical  activities,  23 
Black-leg  vaccine,  preparation  of, 

307 
Bleaching,  237 


Blood,  as  a  medium,  19,  20 

defibrination  of,  302,  323 

dried,  test  of,  322 

enzyme  of,  181 

isolation  of  pathogens  from,  301 
Blood  cells,  red,  injection  of,  324 

cells,  red,  method  of  washing, 
324 

cells,  red,  use  of,  323-329 

serum,  as  a  medium,   19,  20, 
45 

serum,  sterilization  of,  8 
BOHME,  A.,  133 
Boiling,  sterilization  by,  10 
BOLDUAN, 291 

Bombicci's  anaerobic  dish,  159 
Books,  text  and  reference,  list  of, 

381-388 

Botkin's  anaerobic  apparatus,  159 
BOURGEOIS,  32 
Bougie,  12 

Bouillon,  nutrient,  19 
Bread  fermentation,  182 
Bread-making,  yeasts  used,  114 
BREW,  J.  D.,  268 
Broth,  adonit,  362,  363 

carbohydrate,  19 

dextrose,  362,  363 

dulcit,  362,  363 

glycerinated  veal,  303,  304 

lactose,  362,  363 

liver,  362,  363 

neutral  red,  363 

nitrate,  251,  354 

nutrient,  19,  29,  46 

nutrient,  preparation  of,  29-31 

saccharose,  362,  363 

serum,  19 

sterilization  of,  31 

sugar,  19,  84,  358 

sugar-free,  358 

urea,  352 
Brownian  movement,  75 


400 


INDEX 


Buchner's    anaerobic   tube,    161, 

162 

Budding  of  yeasts,  78,  114 
Bullock's    anaerobic     apparatus, 

159 
Butter,  197 

canned,  285 

fat,  preparation  of,  349 

microflora,  284-285 

old,  285 

renovated,  285 

storage,  285 

trier,  use  of,  289,  290 


Caffein,  361 

Calcium  carbonate,  167,  168,  349, 
352,  354,  355     , 

chloride,  351,  351 

citrate,  353 

malate,  353 

phosphate,  di-basic,  263 

phosphate,  mono-basic,  193 

phosphate,  tri-basic,  263,  350 

salts,  action  of,  in  milk,  24 

sulfate,  354 

tartrate,  353 

Calculations      for      microscopic 
counts,  266,  267 

titration,  21,  22 

Calibration  of  filar  micrometer, 
72-74 

lenses,  71-74 
Camphor,  15 

Candles,  filter,  sterilization  of,  6,  7 
Canula,  299 

Capsules,  presence  of,  97,  98' 
Caramelization  of  sugar,  26 
Carbamases,  180 
Carbohydrases,  179 
Carbohydrate,  agar,  37 
Carbohydrates  as  food,  167,  168 

enzymes  of,  179-182 


Carbohydrates,  fermentation  of, 

117,  167 

Carbol-fuchsin,  72,  90,  92,  374 
Carbolic  acid.     See  Acid 
Carbon  bisulfid,  effect  on  pigment, 

175,  176 
Carbon  dioxid,  20,  117-119,  157, 

165,  171,  172,  216,  231,  374 
dioxid.  absorption  of,  117-119 
dioxid,  atmosphere  of,  157 
monoxid,  250 
Carmen  rubrum,  75 
Carrot,  as  a  medium,  20 
Case,  pipette,  17,  18 
Casein,    coagulating   enzyme   of, 

181 

colloidal  state  of,]24,  33 
in  milk,  24 
proteolytic    enzymes    of,    188, 

189 

Caseinase,  181 

Catalase,  181,  184,  191,  239,  240 
Cautery,  6 
Cell  division,  74 

-envelop,  97 
Cellulose,  179 
Cellulose,  decomposition  of,  246- 

249 

enzyme  of,  179 
Centare,  379 
Centigram,  379 
Centimeter,  379 
Centrifuge,  273-275,  323,  324 

tubes,  323 
Ceylon  moss,  37 
Chalk,  369 
CHAMBERLAND,  12,  158,  313,  315 

'filter,  12,  313,  315 
Changes  in  milk,  23-25 
CHAPIN,  C.  V.  222 
Chart,   descriptive.  Soc.  of  Am. 
Bacteriologists,  63,  121,  129, 
132,  226 


INDEX 


401 


Cheese,  cheddar,  286 

microfiora  of,  286,  287 

trier,  286 
Chemical  agents  of  sterilization, 

5,  14,  15 

CHESTER,  F.  D.,  131 
Chinese  ink  preparation,  85,  86 

preparation  of,  372 

use  of,  72,  85,  86 
Chlamydobacteriacece,  348 
Chlamydothrix,  348 
Chloral  hydrate,  use  of,  295 
Chloride  of  lime  in  water  purifica- 
tion, 236-238 
Chlorination,  237 
Chlorine,  available,  236,  237 
Chloroform,  15,  65,  175,  176,  295 
Chlorophyll,    pigment   analogous 

to.  154 

Chromatium,  348 
Chromic  acid   cleaning  solution, 

1,  48,  212 
Chromogenic  organisms,  26,  154, 

264 

Chromoparous  bacteria,  154 
Chromophorus  bacteria,  154 
Chymosin,  181 
Cider,  20,  22,  46,  204 

fermented,  192,  351 

titration  of,  20,  22 
Cladothrix,  348 
Classification  of  nutrient  media, 

18-20 
Classifications,  physiological,  153- 

155 

Clay,  modeling,  165 
Clean,  chemically,  1 
Cleaning  glassware,  1,  2,  3,  4 

powder,  2 

solution,  1,  2,  48,  366 
Cloth,  physicians,  3 
Coagulation,  8,  29,  179 
Cocaine  hydrochloride,  295,  299 


Coccacece,  347 
Cohn's  solution,  172 
Collagen..  32,  38 
Collodion  sac,  13,  299 
Colloid,  reversible,  33,  38,  39 
Colloidal  state  of  casein.  24,  33 
Colloids,  33,  195,  196 
Colonies,  acid,  167,  225,  230 

counting,  56,  57 

zone  development  of,  164 
Colony,  giant,  61-63,  109,  293 

isolated,  58 

mold,  105 

yeast.  120 

formation,  49,  79,  80,  84 
Columella,  100 
Combustion,  total,  184 
Complement,  325-329 

destruction  of,  by  heat,  326 

fixation  test,  323,  325-329 

source  of,  325 

titration  of,  326 
Concentration  of  solutions,  effect 

of,  53,  54 
Condensation  water,  35,  38,  51, 

129,  131 

Condenser,  microscope,  66,  69 
Congo  red  medium,  259 
Conidia,  101 
CONN,  H.  W.,  84,  116,  132,  140, 

144,  147,  177,  243,  287 
Conradi-Drigalski's  agar,  361-363 
Continuous  heating,  8-11 
Contrast  stain,  92,  93 
Cooling,  value  of,  203,  280 
Coprophyl,  154 
Cork  borer,  26 

Cornea,  inoculation  into,  299 
Corpuscles,  red,  method  of  wash- 
ing, 323 

Corrosive  sublimate,  186 
Cotton,  absorbent,  26 

decomposition  of,  248 


402 


INDEX 


Cotton,  effect  of  sterilization  on, 
7 

purpose  of,  15 

waste,  disposal  of,  ix 

plugs,  preparation  of,  16,  17 

plugs,  rolled,  16,  17 
Counterpoise,  30 
Counter-stain,  92,  93 
Counting  colonies,  56,  57 

lens,  56 

plate,  Jeffer's,  56 

plate,  Wolfhiigel's,  56 
Cover-glasses,  cleaning  of,  4 

sterilization  of,  18,  48 
Cow,  aborting,  serum  from,  325 
Cream,  ripened,  microflora  of,  285 

pasteurization  of,  281-284 
Crenothrix,  348 
Cresol,    compound    solution    of, 

14,  48,  296,  297,  365 
Crucible,  Gooch,  as  filter,  13 
Cryophilic  bacteria,  155,  200 
Crystalloids,  33 

Crystals,  formation  of,  176,  252 
Culture,  adhesion,  76,  78-80,  81 

hanging-block,  81,  82 

Lindner's  droplet,  84,  85 

media,  sterilization  of,  8-13 

medium,  31,  37,  49 

methods,  anaerobic,  155-165 
Cultures,  ix,  x,  xi,  44-48 

broken,  48 

care  of,  46-48,  125 

description  of,  129 

dried,  198 

gelatin,  46 

impure,  44,  45,  107 

incubation  of,  46,  47 

liquid,  46,  60 

mixed,  44,  45,  107 

old,  disposal  of,  10,  14,  48 

plate,  45,  49-56 

pure,  44,  45,  49,  107,  114 


Cultures,  shake,  163 

slant,  45 

stab,  46,  61 

stick,  46 

streak,  46,  58,  60 

transferring,  51,  52,  60 
CUMMING,  J.  G.,  13 
Cup,  measuring,  28 
Curd,  acid,  24,  185,  186 

liquefaction  of,  24 

rennet,  24,  185,  186 
Curdling,  cause  of,  24 
Curette,  use  of,  316 
Curves,  plotting,  xi,  112 
Cutaneous  inoculation,  297 
Cuts,  care  of,  48 
Cytase,  179 


Data,  tabulation  of,  xi 
Death-point,  thermal,  200-202 
Decagram,  379 
Decaliter,  379 
Decameter,  379 
Decigram,  379 
Decimeter,  379 
DECKER,  J.  W.,  287 
Decolorization    in    staining,    90- 

93,96 

of  litmus,  23,  24 
Defibrination  of  blood,  302,  320, 

323 

Degrees,  Fuller's  scale,  20,  21 
Denitrification  in   solution,   251, 

252 

studies,  culture  media  for,  354 
Denitrifying  organisms,  19,  251, 

252 
Deodorizer,  chloride  of  lime  as, 

237 

Desiccation,  197 

Desiccator,  as  anaerobic  dish,  160 
Desk,  microorganisms  on,  146, 147 


INDEX 


403 


Dextrin,  179 
Dextrinase,  179 

Dextrose,  23,  156,  170,  180,  182, 
184,  195,  196,  351,  358,  359 
fermentation  of,  180,  182 
influence  on  denitrification,  252 
Dextro-zymase,  180,  182 
Dialysis,   as  a  sterilizing  agent, 

5,  13 

Diastase,  179 
Diatomaceous  earth,  12 
Dichotomous  branching,  100,  101, 

348 

Dilution  flasks,  52,  54 
method,  loop,  49-52 
qualitative,  49-52 
quantitative,  52-56 
straight  needle,  51,  107,  115 
Diphenylamin,  sulfuric  acid  solu- 
tion of,  249,  371 
Directions,  laboratory,  x 
Disaccharides,   enzymes  of,   180, 

182 

Discoloration  in  water,  237 
Discontinuous  heating,  8-10 
Diseases,  animal,  295-329 
plant,  291-294 
producing  bacteria,  153 
Dish,  evaporating,  use  of,  20-22 
Dishes,  deep  culture,  wrapping  of, 

18 

Petri,  wrapping  of,  17,  18 
Disinfectant,  phenol  coefficient  of, 

210-213 
Disinfectants,  ix,  5,  14,  15,  236- 

238,  309 
DISTASO,  157 
Distilled  water,  20 

micron1  ora  of,  152,  153 
DON,  J.  and  CHISHOLM,  J.,  227, 

239 

DORSET,    M.,    MCBRYDE,   C.   N. 
and  NILES,  W.  B.,  315 


Dourine,  diagnosis  of,  329 
Draw-tube  of  microscope,  65 
Drench,  use  of,  300 
Dunham's  solution,    19,   43,   44, 

125,  129,  172 

Durham's  fermentation  tube,  119 
Dust,  microorganisms  in,  146 
Dyes,  acid,  87 

basic,  87 

organic,  reduction  of,  181,  184 

saturated  alcoholic  solution  of, 
87 

stock  solutions  of,  87 

E 

Earth,  diatomaceous,  12 

Egg,  albumin,  liquefaction  of,  24 

as  a  medium,  19,  20 

use  of,  in  preparation  of  media 

30 
Ehrlich's  method  of  testing  indol 

production,  132,  133 
Electrolytes,  53,  195,  196 
Emboli,  from  injection,  299 
Emulsin,  180,  182 
Endo  agar,  361-363 

-enzymes,  178,  190 
Endospores,  114,  121 
End-point,  20 

Enzyme,    carbohydrate-coagulat- 
ing, 181 

action,  pure,  188 
Enzymes,  179-194 

acting  anaerobically,  179 

classification  of,  179-181 

coagulating,  179,  181,  185,  186, 
193,  194 

effect  of  heat  on,  186 

extracellular,  178 

formation  of  name  of,  179 

hydrogen-producing,  23 

hydrolytic,  178,  179,  180,  182- 
185 


404 


INDEX 


Enzymes,  intracellular,  178 

isomer-producing,  179 

lipolytic,  178,  180,  183 

oxidizing,  179,  181,  183,  184 

producing     intramolecular 
change,  178,  180-185 

protein-coagulating,  181 

protein-digesting,  180 

proteolytic,   24,   25,    178,    180, 
187-189 

reducing,  179,  181,  184,  190 

rennet-like,  24 

specific,  167,  169,  172,  190,  192, 
217 

syntheses-producing,  179 
Eosin,  87 
Erepsin,  180 
Ereptase,  180 

ERNST,  WM.,  268,  273,  278,  281 
Esmarch's  tube,  158 
ESTEN,  W.  M.,  132 
Esterases,  180 
Esters,  enzymes  of,  180 

glycerin,  183 
Ether,  6,  92,  175,  176,  295 

flame,  sterilization  in,  6 
Eubacteria,  347,  348 
EULER,  HANS,  15,  168,  179,  190, 

192-194 

Eurythermic  bacteria,  155 
Evaporating  dish,  use  of,  20-22 
Exo-enzymes,  178 
Extracellular  enzymes,  178 
Extractives,  from  meat,  28,  29 
EYRE,  J.  W.  H.,  15,  121,  145,  146, 
158,  159,  161,  167,  175,  198, 
206,  222,  243,  296,  301,  302 


Facultative,  154 

Fat,  butter,  preparation  of,  349 
construction  of,  26,  183 
decomposition  of,  183 


Fat,  enzymes  of,  180, 183 

in  bacteria,  92 

in  milk,  23 
Fats,  natural,  183 
Fermentation,  acetic,  192,  193 

alcoholic,  114 

gaseous,  117-119 

lactic  acid,  167,  184,  217,  218 

Lindner's   method    of    demon- 
strating, 83,  84 

spontaneous,  171 

tube,  Durham's,  119 
Smith's,  117-119 

tubes,  cleaning  of,  4 

tubes,  plugging  of,  18 

urea,  185 

vinegar,  183 
Fermentative,  153 
Ferric  chloride,  214,  354 

sulfate,  351 

Ferrous  sulfate,  350,  353,  375 
Fibrin,    coagulating   enzyme    of, 
181 

liquefaction  of,  24 
Filar  ocular  micrometer,  72-74 
Filter,  Berkefeld,  12,  238,  239 

candles,  sterilization  of,  6,  10 

-paper,  decomposition  of,  246- 
249 

-paper  in  media,  352 

pores  of,  12 
Filters,  bacterial,  12 

Berkefeld,  sterilization  of,  6 

Chamberland,  12 

cleaning  of,  313 

porcelain,  12 

purification  of,  313 
Filterable  organisms,  12,  13 
Filtrate,  germ-free,  12-14 
Filtration,  as  a  sterilizing  agent, 
5,  12,  13,  145,  146 

of  liquid  culture,  210 

rate  of,  13 


INDEX 


405 


FISCHER,  ALFRED,  153,  154,  171, 

178,  197,  200,  203 
FISCHER,  EMIL,  132 
Fish,  salt,  197 
Fishing  a  colony,  59 
Flagella,  93,  94 

staining  method,  93-95,  258 
Flame,  naked,  sterilization  in,  6, 

18,  82,  302 
Flasks,  cleaning  of,  3 
plugging  of,  16,  17 
Roux,  2,  61,  62 
Floor,    microorganisms   on,    146, 

147 

Flowing  steam,  10,  11 
Fluorescent,  360 
Focal  point,  68,  77 
Focusing,  67-69,  77 
Food,  requirements,  variation  in, 

170,  171 

small  amount  needed,  152,  153 
Foods,  microbial,  18-20 
Forceps,  sterilization  of,  6 

use  of,  16 
Form,   for  writing  up  exercises, 

xi,  xii 
Formaldehyde,  188,  213,  214 

ring  test,  214 
Formalin,  14,  188 
Formulae   for  conversion   of   de- 
grees of  temperature,  377 
Freezing,  effect  of,  199,  200 
FROST,  W.  D.,  268 
Fructification  of  molds,  100,  101 
Fruit  juices,  fermented,  20,  22, 

203,   204 

juices,  natural,  20,  22 
juices,  titration  of,  22 
Fruiting  bodies,  79,  100-109 
Fuchsin,  87,  88,  373 
acid,  87 
basic,  373 
FUHRMAN,  F.,  63 


Fuller's  scale,  20,  21,40,  205 
Funnel,  filling,  25 
Furnace,  muffle,  6 

G 

GAGE,  S.  H.,  69 

Galactose,  23,  180,  182 
fermentation  of,  184 

Galacto-zymase,  180 

Garget,  275 

Gas,  absorption  of,  118,  119 

Gasometer,  117,  118,  120 

Gas-producing  bacteria,  154 

Gas  production,  25,  46,  83 
production  in  milk,  25 
production,    qualitative,    117- 
119 

Gauze,  hospital,  3 

Gelatin  as  a  food  material,  32 
constitution  of,  32 
decomposition  products  of,  32 
discussion  of,  31-35 
effect  of  enzymes  on,  180 
effect  of  superdrying  on,  34 
effect  of  superheating  on,  34,  35 
liquefaction  of,  24,  46,  174,  187, 

188,  195-197 
liquefaction,  point  of,  34 
lowering  of  liquefaction  point, 

34,  352,  356 
nutrient,  19,  20,  36,  37 
nutrient,  cooling  of,  36,  52 
nutrient    loss     of     solidifying 

power,  34,  35 

nutrient  preparation  of,  36,  37 
nutrient  sterilization  of,  35-37 
phenol,  187,  188 
physical  properties  of,  32,  33 
salt-free,  356-358 
size  of  molecule,  32 
solidifying  point,  34,  352 
source  of,  32 
acid  in,  34,  187 


:406 


INDEX 


Gelatin,  urea,  352 

waste,  ix 
Gelatinizing    property,    loss    of, 

34,  35 

Gelideum  corneum,  37 
Gemmation,  120 
Gentian  violet,  87 
Germicidal,  1,  365 
Germination  of  mold  spores,  78,  79 

stages  of,  79 
Giant    colony,     preparation    of, 

61-63 
Giltay's  agar,  354 

solution,  19,  251,  252,  354 
GILTNER,  W.,  160-162,  164,  312, 

316 

Giltner's  H  tube,  160-162,  164 
Glanders,  diagnosis  of,  329 
Glands,      lymph,      isolation     of 

pathogens  from,  301 
Glass  rods,  sterilization  of,  6 
Glassware,  cleaning,  1-4 

new,  1 

preparation  of,  for  sterilization, 
15-18 

purpose  of  sterilization,  15,  16 

sterilization  of,  6-15 
Glucosidases,  180,  182 
Glucosides,  enzymes  of,  180,  182 
Glycerin,  26,  183 
Glycogen,  enzyme  of,  179 
Glycogenase,  179 
Gonidia,  348 

Gooch  crucible  as  filter,  13 
Gram,  unit  of  weight,  380 

equivalent,  20 

molecule,  20 

negative,  95-97,  235 

negative  bacteria,  97 

positive,  97,  235 

positive  bacteria,  97 
Gram's  stain,  95-97,  373 
Gram-Weigert  staining  method,  96 


Granules,  metachromatic,  87 
GREEN,  REYNOLDS,  84 
Growth,  rate -of,  109 
GRUZIT,  O.  M.,  361 

GUILLIERMOND,  A.,  182 

Guinea  pigs,  295,  303,  308,  325 
Gypsum,  369 

H 

Hair,  microflora  of,  148,  149,  271 
Halophile,  154,  351 
HAMILTON,  H.  G.  and  OHNO,  T., 

213 

HAMMER,  B.  W.,  291 
Hands,  bacteria  on,  148,  272 

sterilizing  of,  14 
Hanging  block,  agar,  81,  82 
Hanging  drop,  59,  74-77 

purpose  of,  74 
HANSEN,  E.,  121 
Haplobacterince,' 347 ,  348 
HASTINGS,  E.  G.,  189 
HAWK,  P.  B.,  172,  190,  214 
Hay,  bacteria  on,  271 

infusion,  9,  19 
Hazen  theorem,  238 
HEADDEN,  W.  P.,  255 
Heat,  as  sterilizing  agent,  5-12 
Heat,  dry,  202 

for  sterilizing,  5-7,  16 

moist,  5,  8-12,  202 
Heating,  continuous,  8-11 

discontinuous,  8-10 

intermittent,  8-10 
Heat-producing  bacteria,  154 
Hectare,  379 
Hectogram,  379 
Hectoliter,  379 
Hectometer,  379 
Hemicelluloses,  enzymes  of,  179 
Hemoglobin,  28 
Hemolysin,  325-329 

preservation  of,  326 


INDEX 


407 


Hemolysin,  source  of,  325 

titration  of,  327 
Hemolysis,  325-329 
Hemolytic  serum,  preparation  of, 

323,  324 
system,  328,  329 

Hesse's  method  for  anaerobes,  157 
Hexoses,  23,  180 
HILL,  81 
Hoffman  and  Fiske's  enrichment 

medium,  361,  362 
HOFFMAN  C.  and  HAMMER,  B.  W., 

255 

HOFMEISTER,  32 

HOLM,  M.  L.  and  GARDNER,  E.  A., 

213 

HOOKER,  A.  H.,  236-238 
Hospital  gauze,  3 
Hot  air  sterilization,  7 
HUEPPE,  38 
Humus,  239,  240 

Hydrochloric  acid,  N/l,  prepara- 
tion ot,  367,  368 
acid  N/20,  20 
acid  N/20,  preparation  of,  367, 

368 

Hydrogel,  33 
Hydrogen,    atmosphere    of,    157, 

164,  165 
peroxide,  enzyme  of,  181,  184, 

191,  239,  240 

sulfid,  Beijerinck's  test  for,  174 
sulfid,  production  of,  174,  175, 

190,  191 

sulfid,  test  for,  130 
Hydrophobia,  13 
Hyphse,  100,  101,  108 


IAGO,  WM.  and  IAGO,  WM.  C.,  116 
Ice  cream,  microflora  of,  289-291 
Illumination  for  microscope,  67,  69 
Image,  reversed,  in  microscope,  68 


Immunity,  295,  301,  303-329,  370 
in  plants,  294 
production  of,  295 
tests,  13 

Impression   preparation,   98,   99, 
375 

Inactivated  serum,  325-329 

Inactivation  of  serum,  326,  328 

Incision,  crucial,  299 

Incisions,  297 

Incubator,  37°,  53 

India  ink,  85 

Indicators,  20,  369 

Indigo,  181 

Indol,  43,  131-133,  370 
Ehrlich's  test  for,  132,  133 
graphic  formula  of,  132 
in  peptone,  132 
solutions,  370 
test  for,  132,  133,  370 

Infection,  x 

Infusion,  hay,  9,  19 

meat,  preparation  of,  28,  29 

Inhibit,  1 

Inhibitive  action,  216 

Ink,  Chinese,  85,  86,  243,  247 
preparation  of,  372 
preparation,  Chinese,  85,  86 

Inoculation,  animal,  295-300 
cutaneous,  method  of,  297 
ingestion,  method  of,  300 
intraabdominal,  method  of,  299 
intramuscular,  method  of,  298 
intraorbital,  method  of,  299 
intraperitoneal,  method  of,  299 
intrapulmonary,  method  of,  300 
intravenous,  method  of,  298 
of  media,  45,  46,  59-61 
subcutaneous,  method  of,  297 
subdural,  299 

Inoculations,  mold,  62 

Inoculum,  14,  197,  297 

Inosit,  359 


408 


INDEX 


Instruments,   metal,   sterilization 

of,  6,  14,  15 
Intermittent   heating,    8-10,    37 

139 
Intraabdominal  inoculation,  29£ 

322 

Intracellular  enzymes,  178 
Intramolecular  change,  178,  180 

182,  184,  185 

Intramuscular  inoculation,  298 
Intraorbital  inoculation,  299 
Intraperitoneal  inoculation,  299 
Intrapulmonary  inoculation,  300 
Intravenous  inoculation,  298 
Inulin,  363 

Inversion  of  sugars,  23 
Invertase,  180 
Invertin,  180 
lodin   solution,   Lugol's,  95,   96, 

114,  115,  189,  309,  375 
tincture  of,  14,  365 
Iris  diaphragm,  63-69 
Iron,  citrate,  soluble,  360 

sulfate,  350 
Isinglass,  Bengal,  37 
Isolation    of   pure    cultures,    31, 

52-56 
Itch,  barber's,  149 


Jaffna  moss,  37 
Jenner's  stain,  320 
JENSEN,  C.  O.,  273,  278 
JONES,  DAN  H.,  255 
JONES,  L.  R.,  294 
JORDAN,  E.  O.,  31,  132,  145,  199, 
202,  207,  303 

JOUBERT,  158 
JUNGANO,  157 

K 

Kidney,    isolation    of    pathogens 
from,  301,  302 


Kieselguhr,  12 

Kilogram,  379 

Kiloliter,  379 

Kilometer,  379 

Kipp    generator    for     hydrogen, 

165 

Kitasato's  dish,  159 
Klatschpraparat,  98 
KLOCKER,  A.,  101,  114 
Knife,  potato,  26 
Knives,  sterilization  of,  6 
KOCH,  ROBERT,  31,  32 
Koch's  first  plates,  31,  32 
KRUSE,  W.,  191 


Labeling  plates,  52 

Lacomme's  tube,  158 

Lactacidase,  181,  184 

Lactase,  180,  182 

Lactic  acid,  23,  184 

Lactose,  23,   167,   170,  223,  356 

enzyme  of,  180,  182 

fermentation  of,  182,  184 
LAFAR,  F.,  63,  78,  84,  101,  112, 
121,  167,  169,  172,  173,  177, 
191,  193,  199,  200,  203,  205, 
209,  217,  262 
Lamprocystis,  348 
Laparotomy,  299 
LASER,  169 
Lead  acetate,  165 

acetate     paper,    use    of,    125, 
190 

carbonate,  174 

Leguminosce,  nodules  of,  256-263 
LEHMANN,  K.  B.  und  NEUMANN, 

R.  O.,  63 

Leuco-compound  of  litmus,  23 
Leucocytes,  299,  319,  320 

in  milk,  275 
Level,  spirit,  50 
Leveling  stand,  50 


INDEX 


409 


Levulose,  180 

fermentation  of,  180 
Levulozymase,  180 
Liborius-Veillon  anaerobic  meth- 
od, 163 

Lichens,  as  source  of  litmus,  369 
Light,  artificial,  209 

artificial  for  microscopical  work, 

67 

diffused,  206,  207 
producing   bacteria,    154,    177, 

178 
relation  to  pigment  formation, 

176 
Lindner's   concave  slide  culture, 

120 

droplet  culture,  84 
fermentation  method,  83,  84 
Lipase,  180,  183 
Lipases,  action  of,  183 
LIPMAN,  J.  G.  and  BROWN,  P.  E., 
244,  247,  249,  251,  253,  255, 
263,  354 
Liquefaction  of  boiled  egg  white, 

24 

of  fibrin,  24 

of  gelatin,  24,  46,  187,  188 
of  milk  curd,  24 
Liquor    cresolis    compositus,    48, 

296,  297,  365 
Liter,  379 
Litmus,  decolorization  of,  23,  25, 

181 

Merck's  purified,  369 
milk,  23-26 

reduction  of,  23,  24,  181 
solution,  23,  26 
solution,  purpose  of  369 
source  of,  369 
Loam,  clay,  Azotobacter  in,  253 

sandy,  Azotobacter  in,  253 
Loeffler's  alkaline  methylen  blue, 
265 


LOHNIS,  F.,  63,  133,  168,  173,  175, 
192,  241,  244,  247,  249,  251, 
253,  255,  262,  264,  268,  278, 
285,  287 

Longevity,  198 

Loop  dilution  method,  51 

Lugol's  iodin  solution,  95,  96,  114, 
115,  189,  309,  375 

M 

Macroscopical  changes,  23,  142 
Magnesium      ammonium      phos- 
phate, 246 

carbonate,  basic,  353 
oxid,  245 
phosphate,  252 
sulfate,  350-355 
Magnification,  70-74 
Maltase,  180,  182 
Malt  extract,  19 
Maltose,  enzyme  of,  180,  182 
fermentation  of,  182 
from  starch,  182 
Manganese  sulfate,  351 
Mannit,  fermentation  of,  253,  254 

solution,  253-255,  354 
Manure,    anaerobic    bacteria   in, 

165 

Azotobacter  in,  253 
bacteria  in,  165,  241,  243,  248, 

253 

cellulose-decomposing      organ- 
isms in,  246,  247 
Measuring    microorganisms,    70- 

74,  109 

MARSHALL,  C.  E.,  15,  101,  113, 
114,  140,  141,  144-147,  149, 
151,  153,  167,  169,  171-173, 
175,  178,  186,  187-193,  197- 
200,  202,  203,  205-207,  209, 
213-215,  217,  219,  222,  227, 
233,  239,  243,  244,  248,  249, 
251,  253,  255,  262,  264,  268, 


410 


INDEX 


270,  273,  278,  281,  285,  287, 
289,  291,  294,  303,  305,  307, 
308,  310,  312,  323,  331 
Mason  jar,  160 
McBETH,  I.  B.  and  SCALES,  F.  M., 

249 

McBRYDE,  C.  N.,  315 
MCCAMPBELL,  E.  F.,  318,  321 
MCFARLAND,  J.,  91,  312,  321,  323 
McLeod's  plate  base,  163 
Mastitis,  275 
Meat,  as"a  medium,  19 
infusion,  preparation  of,  28,  29 
products,  as  media,  19 
Media,  acid,  22 
albuminous,  8 
alkaline,  20 

culture,  sterilization  of,  8-13 
liquid,  19,  20,  22-26 
mounting,  372 
natural,  19,  20,  22-26 
nutrient,  19,  20,  22-26,  44 
nutrient,  classification  of,  19,  20 
over-heating  of,  26 
prepared,  19,  20 
solid,  19,  20 
solid  liquefiable,  20 
solid  liquefiable,  disposal  of,  ix 
solid,  nbn-liquefiable,  20 
synthetic,  19,  20,  40,  170,  171 
water  analysis,  356-361 
Medium,   culture,   solid,   31,   37, 

45,46 
enrichment,       Hoffman       and 

Fiske's,  361,  362 
glycerin  potato,  26,  27,  45 
liquefiable  solid,  46 
physical  condition  of,  131 
standard  liquid,  29 
Membrane,  semi-permeable,  13 
Mercuric  chloride,  364 
corrosive  character  of,  364 
germicidal  action  of,  364 


Mercuric  chloride,  poisonous  na- 
ture of,  364 
solubility  of,  364 
stock  solution,  364 
synonyms  of,  364 
1-500,  210,  212,  259 
1-1000,  x,  1,    14,  48,  77,   147, 

210,  212,  268 
Mesophilic  bacteria,  155 
Metabiosis,  45,  204,  215-217 
Metatrophic,  154,  155 
Meter,  379 
Methylen  blue,   23,   87-93,    179, 

181,  184 

enzyme  of,  179,  181,  184 
for  yeasts,  373 
Loeffler's    alkaline,    265,     266, 

374 

leuco-base  of,  184 
reductase,  181,  184 
Methyl  indol,  132 
Metric  system,  379,  380 
Mice,  white,  295 
Micrococcm  gonorrhea,  97 
tetragenus,  97,  347 
varians,  195,  196 
Microflora  of  hair,  148,  149 
of  the  mucous  membrane,  150, 

151 

of  skin,  148 
of  soil,  241-244 
Micrometer,  head,  65 
object,  70-74 
ocular,  70-74,  265 
ocular,  filar,  72-74 
stage,  70-74,  265 
step,  70,  71,  74 
Micromillimeter,  379 
Micron,  33,  70;  71,  73,  74,  379 
Microorganisms,  arrangement  of, 

74 

chromogenic,  26 
determination  of  size,  70-74 


INDEX 


411 


Microorganisms,  effect  of  sunlight 
on,  208,  209 

filterable,  12,  13 

food  requirements  of,  18,  19 

in  soil,  241-244 

measuring  of,  70-74 

pathogenic,  26,  301,  303,  308, 
310,  313,  315 

products  of,  15 

rennet-producing,  24 

resistance  of,  10 

salt-resisting,  197 

starch-digesting,  189,  190 

sugar-resisting,  197 
Microscope,  ix,  58,  63-69 

carrying  the,  65 

cleaning  the,  65,  66 

focusing  the,  65-69 

how  to  use,  63-69 

lighting  for,  67 
Microspira  comma,  347 

deneke,  97,  347 

finkler  prior,  97,  347 
MIGULA,  W.,  347,  348 
Migula's  classification,  modified, 

347,  348 
Milk,  acid  production  in,  23 

action  of  calcium  salts  in,  24 

as  a  medium,   19,   20,   22-26, 
46,  129,  141-144 

"  bloody,"  177 

blue,  177 

bottles,  bacteria  in,  272 

cans,  bacteria  in,  272 

cells  in)  267 

changes  in,  23-25 

clarifier,  value  of,  276 

composition  of,  23 

condensed,  microflora  of,  288, 
289 

cow's,  coagulating  enzyme  of, 
181 

curd  formation  in  24,  280 


Milk,  dilutions  for  plating,  142, 
264,  268,  269,  274,  276,  279, 
280,  282 

dirt  in,  273-278 

effect  of  straining,  276-278 

extraneous    contamination    of, 
270-273 

fore,  bacteria  in,  268,  269 

gas  production  in,  25 

germicidal  action  of,  280 

human,  coagulating  enzyme  of, 
181 

keeping  quality  of,  278-281 

litmus,  23,  25,  26,  361,  363 

litmus,  preparation  of,  25,  26 

market,  278-281 

microflora  of,  264-268,  274 

middle,  bacteria  in,  268,  269 

pails,  bacteria  in,  272 

pasteurization  of,  203-205,  281- 
284 

peptonization  of,  24,  25 

phenol,  185,186 

pure,  278-281 

reaction  of,  25,  177 

red,  177 

separated,  25 

skimmed,  25 

sour,  25 

staining  of,  96 

standards,  bacteriological,  268 

sterilization  of,  26,  139 

strippings,     bacteria    in,     268, 
269 

titration  of,  20-23 

viscosity  of,  283,  288 

whole,  25 
Milking  tube,  269 
Millier,  380 
Milligram,  380 

equivalent,  20 
Millimeter,  33,  63,  70,  379 
Millimicron,  33,  379 


412 


INDEX 


Mirror,  concave,  67 

plane,  67 
MOHLER,  J.  R.  and  EICHHORN,  A., 

329 
MOHLER,  J.  R.,  EICHHORN,  A.  and 

BUCK,  J.  M.,  329 
Moist   chamber,   preparation   of, 

80,81 

culture,  100,  109 
Moisture, 

relation   to  microbial  destruc- 
tion by  heat,  202,  203 
Mold  colony,  examination  of,  105, 

107,  109 
growth,  characteristics  of,  100- 

104,  106,  108 

spores,  germination  of,  78-80 
Molds,  100-113,  182 
in  air,  221 

influence  of  light  on,  206,  207 
microscopical    examination   of, 

105 

pathogenic  nature  of,  113 
resistance  to  heat,  202 
study  of,  107-111 
MOORE,  V.  A.,  156,  302,  305 
MOORE,  V.  A.  and  FITCH,  C.  P., 

301,  302 
Mordant  for  flagella  staining,  94, 

375 
MORTENSEN,  M.  and  GORDON,  J., 

291 
Moss,  Ceylon,  37 

Jaffna,  37 

Motility,  74,  75,  86,  129 
Mounts,  permanent,  372 
Mouth,  microflora  of,  150,  151 
Mouths  of  culture  tubes,  flasks, 

sterilization  of,  6 
Movement,  Brownian,  75 
molecular,  75 
rate  of,  74 
Mucilage  of  Ps.  radicicola,  258 


Mucor,  63,  100 

stolonifer,  100 
Mucous  membrane,  microflora  of, 

150,  151 

Mucus,  staining  of,  87 
Muffle  furnace,  6,  7 
MUIR,  R.  and  RITCHIE,  J.,  163 
Mutual  relationship,  215-217 
Mycelium,  79,  100-102,  105,  107- 

110,  112,  113 

Mycoderma,  169,  184,  195,  196 
Mycodermata,  114 
Myriagram,  380 
Myriameter,  379 

N 
a-naphthylamin,    133,   249,    251, 

371 
Needles,    platinum,    sterilization 

of,  6 

Nephelometer,  319 
Nessler's  solution,  133,  249,  251, 

371,  372 

Neutralization,  20,  22,  25 
Newspaper,  uses  of,  17,  18 
Nitrate  peptone  solution,  19,  44 

reductase,  181 

Nitrates,  reduction  of,  44,  131, 
133,  134,  138,  181,  251-253, 
363 

test  solutions  for,  371 
Nitrification,  255 
in  solution,  249-251 
studies,  media  for,  353,  354 
Nitrifying  bacteria,  153,  353 
Nitrite  reductase,  181 
Nitrites,  43,  44, 133, 134, 138,  250, 

252 

reduction  of,  181 
test  solutions  for,  371 
Nitrogen  as  food,  172,  173 
atmosphere  of,  157,  165 
cycle,  255 


INDEX 


413 


Nitrogen-fixation  studies,   media 

for,  354,  355 
-fixation,    non-symbiotic,   253- 

255,  264 
-fixation,    symbiotic,    256-263, 

264 

from  nitrates,  44,  134 
inorganic  as  food,  172,  173 
organic,  172,  173 
Nodule  formation,  observation  of, 

259-262 
Nodules,   on  leguminous  plants, 

256-263 

Non-electrolytes,  195,  196 
NORGAARD,  V.  A.  and  MOHLER, 

J.  R.,  307 

Normal  acid,  20-22,  367,  368 
alkali,>0-22,  368 
solutions,  20-22,  367,  368 
NORTHRUP,  Z.,  169,  215 
Nose,  microflora  of,  150 
Nosepiece,  collar,  66 

triple,  65 
Notebook,  xi 
NOVY,  F.  G.,  132,  158,  160,  164, 

202 
Novy's  jars,  anaerobic,  158,  160, 

164 

NOYES,  WM.  A.,  20 
Nuclei,  stain  for,  99 
Nutrient  media,  18-20,  23-44 
Nutrose,  361 
NUTTALL,  G.  H.  F.,  323 

O 

Objective,  oil  immersion,  69,  71 
Objectives,  63,  66,  67,  69,  71,  77 

achromatic,  71 

cleaning  of,  66 
Obligate,  154 
Oculars,  66-72 

cleaning  of,  66 
Odor,  fecal,  cause  of,  132 


Oidiam,  101 
Oil,  cedar  wood,  372 
immersion,  66,  69,  372 
linseed,  365 
of  garlic,  15 
of  mustard,  15 
olive,  157 

Oleomargarine,  285 
Omelianski's  medium,  248,  352 
Oospora  lactis,  101,  107,  108,  169, 

198,  205,  207,  214,  215 
Operation,  site  of,  297-300 
Opsonic  index,  determination  of, 

319-321 

test,    McCampbelPs   modifica- 
tion, 321 
Opsonins,  demonstration  of,  319- 

321,370 
Optical  axis,  66 
Orcin,  369 
Organic  matter,  effect  of  chloride 

of  lime  on,  236-238 
OBI,  157 

Osmotic  pressure,  52-54,  196 
Outline,  for  study  of  microbiology, 

331-346 

Oven,  for  hot-air  sterilization,  7 
Over-heating  media,  26 
Oxidases,  181,  183,  184 
Oxidation,  183,  184,  237 
Oxygen,  absorption  of,  156,  160- 

164 

free,  liberation  of,  181,  184,  240 
requirements,  131 
tolerance,  154,  164 
transference  of,  181 


Paper,  effect  of  sterilization  on, 

7 

filter,  in  media,  352 
lens,  66 
uses  of,  15,  16 


414 


INDEX 


Papilionacece,    attacked    by    Ps. 

radicicola,  256 
Parachrome  bacteria,  155 
Parachymosin,  181 
Para  -  dimethyl  -  amido  -  ben- 

zaldehyde,  133,  370 
Paraffin,  76,  78,  80 

oil,  157,  308 
Paraformaldehyde,  214 
Parasite,  wound,  293 
Parasites,  facultative,  154 

obligate,  154 
Paratrophic,  154 
Paratyphoid  group,  362 
Parfocal,  66 
PARUMBARU,  38 
PASTEUR,  L.,  8,  13,  158,  159 
Pasteurization,  8,   203-205,   215, 

281-284 
effect  on  digestibility  of  milk, 

283 
factors    influencing    efficiency, 

283 

Pathogenicity,  295 
Pathogenic  material,  care  of,  x 
nature  of  molds,  113 
organisms,  26,  88,  129,  151,  153 
organisms,  care  in  staining,  88 
PAYEN,  38 

Peat,  bacteria  in,  242,  243 
Pecilothermic  bacteria,  155,  200 
NPectase,  180 
"Pectin,  181 
Pectinase,  181,  294 
Pectose,  enzyme  of,  180 
Pencillium,  63 
italicum,  101, 104, 105, 107, 195, 

196,  207 

Pentoses,  enzymes  of,  180 
Pepsase,  180 
Pepsin,  24,  180 
Peptase,  180 
Peptone,  29,  43,  44, 170, 172 


Peptones,  enzymes  of,  180 
Peptonization,  24 

of  milk,  24 
Perhydridase,  181 
Pericardial     fluid,     isolation     of 

pathogens  from,  302 
Periodicals,  list  of,  388,  389 
Peroxidase,  181,  191 
Petri  dishes,  cleaning  of,  3 

wrapping  of,  15,  17,  18 
Phenol  (see  carbolic  acid) 
coefficient,  210-213 
remedy  for  burns  caused  by,  365 
stock  solution  of,  364 
synonyms  of,  364 
value  as  a  disinfectant,  365 
Phenolphthalein  as  indicator,  20- 

22,  369 
Phosphates,  insoluble  to  soluble, 

263,  264 

Phosphorescence,  177,  178 
Phosphorus,    relation    to    decay, 

263,  264 

Photogenic,  154,  177,  178,  351 
Phototaxis,  206,  207 
Phototropism,  207 
Phragmidiothrix,  348 
Physical  agents  of  sterilization,  5 

condition  of  medium,  198 
Physician's  cloth,  3 
Physiological  efficiency,  262,  293 
Physiology     of     microorganisms, 

152-219 

Pickles,  brine,  197 
Pigment,  crystals  of,  176 
formation,  effect  of  temperature 

on,  175 
formation,    relation   of  air  to, 

175 

formation,  relation  of  light,  176 
-producing  bacteria,   154,   155. 

175-177 
solubility  of,  176 


INDEX 


415 


Pigments,     microbial,    effect     of 
physical  and  chemical  agen- 
cies on,  175-177 
Pipette  case,  17,  18 
Pipettes,  cleaning  of,  3 

preparation    of,    for    steriliza- 
tion, 18 

use  of,  22,  54,  55,  112 
Planococcus  agilis,  347 
Planosarcina  mobilis,  347 
Plants,     microbial     diseases     of, 

291-294 
Plasmolysis,  54 
Plasmoptysis,  54 
Plates,  agar,  51,  52 
gelatin,  52 
inverting  of,  52 
Plating,  45,  49-56 

room,  81 
Platinum,   spongy  for  absorbing 

oxygen,  157 

needles,  sterilization  of,  6 
Pleuritic  fluid,  isolation  of  patho- 
gens from  302 
Plugs,  cotton,  15-17 

rolled,  16 

Poikilothermic  bacteria,  155 
Polypeptids,  enzymes  of,  180 
Folysaccharides,  enzymes  of,  179, 

180,  182,  184 
Porcelain  niters,  12,  13 
Pores  of  filter,  12 
Pork,  salt,  type  of  rrJcroflora,  197 
Potability  of  water,  227 
Potassium  dichromate,  366 
hydroxide,  134,  365,  374 
iodid,  365,  375 
nitrate  as  food,  44,  252,  354 
permanganate  solution,  use  of, 

313 

persulfate,  133,  370 
phosphate,   di-basic,   170,  246, 
247,  350-356 


Potassium  phosphate,  mono-basic, 

350,  354 
Potato,  as  a  medium,  19,  20 

glycerin,  26,  27,  45 

knife,  26 

tubes,  preparation  for  steriliza- 
tion, 26 

tubes,  Roux,  26 
Precipitin  test,  321-323 
Precipitate,  flocculent,  in  media,  40 
PRERCOTT,  S.  C.  and  WINSLOW,  C. 

E.  A.,  153,227,228,233,360 
Pressure,  for  filtration,  13 

osmotic,  53,  54 

steam  under,  10,  12 

temperature  table,  376 
Probe,  use  of,  297 
Products,  metabolic,  217-219 

microbial,  study  of,  15,  217-219 
Protamins,  180 
Protease,  180 
Protein  decomposition  products, 

132 

Proteinases,  180 
Proteins,  enzymes  of,  180 

soluble,  as  food,  172,  173 
Proteolytic  enzymes,  24,  180, 185- 

189 

Proteoses,  enzymes  of,  180 
Proteus  vulgaris,  97,  352 
Protoplasm,  stain  for,  87 
Prototrophic,  154 
Protozoa,  140,  141 
Pseudomonas  campestris,  97,  208, 
347 

lucifera,  177 

medicaginis,  97 

pyocyanea,  175 

radidcola,  173,  256-263,  355 

radicicola,    isolation  from    no- 
dule, 256-263 

radicicola,     physiological     effi- 
ciency of,  256-263 


416 


INDEX 


Psychrophilic  bacteria,  155 
Ptyalin,  179 
Pumice  stone,  2 

Pump,      electric      vacuum      or 
pressure,  159 

mercury  vacuum,  159 

water  vacuum,  159 
Purification  of  filter  candles,  313 
Pus,  collecting,  14 

isolation  of  bacteria  from,  148, 
149 

staining  of,  87,  92 
Putrefactive,  153 


Quintol,  380 


R 


Rabbits,  295,  309,  322-325 

Rabies,  13 

RAHN,  O.,  168,  219,  246 

Rats,  white,  295 

Rays,  light,  209 

radium,  209 

X,  209 

Reaction,  20-22,  29,  30,  36,  112, 
199,  206,  215 

adjustment  of  21,  30,  36 

amphoteric,  of  casein,  24 

maximum,  206 

minimum,  206 

of  water  analysis  media,  357 

optimum,  29,  206 
Reactions,  enzymic,  182-185 
Reductase,  methylen  blue,   181, 
184 

nitrate,  181 

nitrite,  181 

sulfur,  181 
Reductases,  181,  184 
Reduction,  of  litmus,  23,  24 

organic  dyes,  181,  184 
References,  xii,  381-389 


Refraction,  counteracted,  372 

index  of,  69,  372 
Refractivity  of  spores,  131 
Refrigerator,  use  of,  28 
Rennet,  24,  181,  193,  194 

curd,  24 
Rennin,  181 

Resistance  of  microorganisms,  10, 
27 

spores,  10 

Respiration  of  bacteria,  165,  177 
DE  REYPAILHADE,  J.,  190 
Rhabdochromalium,  348 
Rhizopus  nigricans,  100,  102,  103, 

207 

Rhodobacteriacece,  348 
RICHMOND,  H.  D.,  194 
RIDEAL,  S.  and  RIDEAL,  E.  K., 

213 

Ringworm,  149 
ROGERS,  L.  A.',  285 
Root  tissues,  invasion  by  bacteria, 

291-294 

ROSENAU,  M.  J.,  270,  278,  284 
Rot,  soft,  bacterial,  291-294 
Roux,  309 
Roux's  anaerobic  tubes,  158 

biological  method  for  anaerobes, 
163,  164 

flask,  62,  109 

tubes  for  potatoes,  26 
Rubber  apparatus,  sterilization  of 

9 

RTJEHLE,  G.  L.  A.,  221,  222 
Ruffer's  flask,  159 
Rules  for  culture  media,  19 

laboratory,  ix,  x 

RUSSELL,  H.   L.  and  HASTINGS, 
E.  G.,  205,  270,  281,  285,  287 

S 

Sac,  collodion,  13 
conjunctival,  295 


INDEX 


417 


Saccharomyces,  apiculatus,  83, 114, 

120 
cerevisice,  83,  97,  114,  115,  120, 

171,  182,  195,  198,  199 
ellipsoideus,  114,  173,  215,  216, 

351 

fragilis,  182 
kefir,  182 

membrancefaciens,  114 
tyricola,  182 
Saccharomycetes,  114 
Saccharophile,  154 
Saccharose,    170,    171,    195,   355, 

356,  359 

enzyme  of,  180,  182 
fermentation  of,  182 
SADTLER,  S.  P.,  172,  190,  193,  289 
Salkowski-  Kitasato  test  for  indol, 

132 
Salt,  citrated,  370 

effect  on  microorganisms,  196, 

197 

normal,  370 
phenol,  303,  304 
physiological,  370 
purpose  of,  in  media,  29 
solution,  52,  370 
solutions,  370 
Salts,  soluble,  of  meat,  28 
Sand  grains,  size  of,  245,  246 

quartz,  245 

Sapo  mollis  U.  S.  P.,  365 
Saprogenic,  153 
Saprophile,  154 
Saprophyte,  154 
Rartina  lutea,  175,  347 
SAVAGE,  W.  G.,  214,  227,  233,  268, 

273,  287,  289,  291 
SAYER,    W.    S.,    RAHN,    O.    and 

FARRAND,  BELL,  285 
Scale,  Fuller's,  20,  21 
Scarification,  for  inoculation,  297 
Schardinger's  reaction,  181 


SCHULTZ,  N.  K,  41 

SCHUTZENBERGER,  32 

Sealing  anaerobic  cultivations,  156 

microscopical  preparations,  76, 

79,  80,  82,  83;  85 
Seaweeds,  agar  from,  37 
Sediment  test,  macroscopic,  276- 
278 

microscopic,  274-276 

tester  for  milk,  274-278 
Sedimentation  tubes,  274 
Seed  inoculation,  259-263 
Seeds,  sterilization  of,  259-261 
Selective  action,  170 
Septate,  100,  101 
Serum,  blood,  310 

blood  sterilization  of,  8 

hemolytic,  production  of,  323, 
324 

hemolytic,  titration  of,  326,  327 

immune,  311 

inactivation  of,  325,  328 

suspect,  325,  328 
Serums,  preservation  of,  15,  328 
Sewage,    bacteriological    analysis 
of,  223,  228-233 

purification  of,  236-239 
Sheaths  of  bacterial  cells,  348 
Sheep,  use  of,  325-329 
Silicic  acid,  colloidal  state  of,  33 
Silver  nitrate  solution,  165 
Size    of    microorganisms,    deter- 
mination of,  70-74 
Skatol,  132 
Skin,  microflora  of,  148,  149,  272 

sterilization  of,  14 
Slides,  cleaning  of,  4 

labelling  of,  89 

permanent,  89 

sterilization  of,  18,  48 
Slime-forming  organisms,  97,  197, 

202 
SMIRNOW,  M.  R.,  27 


418 


INDEX 


SMITH,  ERWIN  F.,  41,  113,  198, 

209,  263,  294 
SMITH,  THEOBALD,  117 
Smith's  fermentation  tube,  117 
Soap,  linseed  oil  potash,  305 
Sodium  carbonate,  26,  351,  353, 

367 

chloride,  350-354,  370^ 
citrate,  319,  37& 
formate,  156 

hydroxide,    effect    of,    on   pig- 
ment, 175,  176 
hydroxide  for  anaerobic  culture, 

156,  367 
hydroxide   for    cleaning,   3,   4, 

212,  367 
hydroxide  for  CO2  absorption, 

367 

hydroxide  N/l,  21,  22,  29,  368 
hydroxide  N/20,  20,  21,  368 
lactate,  356 
nitrate,  170,  354 
nitrite,  353 
sulfindigotate,  156 
taurocholate,  360 
Sohngen's  solution,  353 
Soil,  as  a  medium,  19 
borer,  241 

catalytic  power  of,  239-241 
cellulose-decomposing    bacteria 

in,  246-249 
denitrifying  bacteria   in,    251- 

253 

diseased  spots  in,  255 
extract,  preparation  of,  351 
microscopical    enumeration    of 

bacteria  in,  243,  244 
nitrifying  bacteria  in,  249-251 
Solution,  albuminoid-free,  353 
citrated  salt,  319 
Cohn's,  350 
culture  for  nitrifying  bacteria, 

353,  354 


Solution,  Giltay's,  354 

mannit,  253,  254,  354,  355 

Nessler's,  133,  134,  371,  372 

Sohngen's,  353 

Uschinsky's,  350 

Solutions,  concentration  of,  52-54, 
195-197 

normal,  20-22,  367,  368 

standard,  367,  368 
Spatulas,  iron  and  nickel — steril- 
ization, 6 

Specific  gravity,  alcohol  by  vol- 
ume, 377 
Spectrum,  209 
Spirillacece,  347 
Spirillum  rubrum,  348 
Spirocheta  obermeieri,  87,  348 
Spirosoma  nasale,  347 
Spleen,    isolation    of    pathogens 

from,  301 

Sporangiophore,  100,  102,  103 
Sporangium,  100,  102,  103 
Spore  formation,  study  of,   144, 

145 

Spores,  ix,  5,   74,   100-104,   107, 
109,  110,  144-145 

bacterial    destruction    of,     by 
heat,  9,  10,  26,  27 

effect  of  sunlight  on,  209 

mold,  germination  of,  78,  85 

resistance  of,  10,  35,  144,  145, 

197-202 

Spore  stain,  90,  91 
Sputum,  staining  of,  92,  93 
Staining,  58,  87-89 

capsules,  97,  98 

flagella,  93-95 

Gram's  method,  95,  97 

Gram-Weigert  method,  96 

in  mvo,  116 

of  tissues,  96 

purpose  of,  87 

time  necessary'  for,  88 


INDEX 


419 


Stains,   anilin-water,   72,  95,  96, 

373 

aqueous-alcoholic,  72,  373 
preparation  of,  373,  374 
saturated  alcoholic,  72,  373 
Stand,  microscope,  63,  65 
Staphylococcus  pyogenes  albus,  97, 

318,  347 

aureus.  97,  318,  347 
Starch,    decomposition    of,    182, 

189,  248 

enzymes  of,  179,  182,  189 
grains,  in  yeast  cake,  116 
soluble,  179,  189 
Steam,  flowing,  10 
high  pressure,  10,  11 
sterilization,  10,  12 
superheated,  10,  11 
Steapsin,  180 

Stearin,  enzyme  of,  180,  183 
Stearinase,  180,  183 
Stenothermic  bacteria,  155 
Sterigmata,  101 
Sterilization,  5-15 
by  antiseptics,  15 
by  continuous  or  discontinuous 
heating  in  water  at  100°  C., 
8,9 

by  dialysis,  13 
by  disinfectants,  14,  15 
by  dry  heat,  6,  7 
by  dry  heat  in  ether  flame,  6 
by  dry  heat  in  hot  air,  6,  7 
by  dry  heat  in  muffle  furnace,  6 
by  dry  heat  in  naked  flame,  6 
by  filtration,  12,  13 
by  flowing  steam  at  100°  C.; 
continuous  or  discontinuous, 
10,  139 

by  moist  heat,   continuous  or 
discontinuous  heating  at  low 
temperatures,  8 
fractional,  34,  139 


Sterilization   of.  a  large  bulk   of 
medium,  26 

of  agar,  43 

of  litmus  milk,  26 

of  meat  infusion,  28 

of  nutrient  broth,  31 

of  nutrient  gelatin,  37 

of  potato  medium,  27 

of  water  analysis  media,  356 

superheated  steam,  11 

Tyndall  method,  8,  9,  139 
Sterilizer,  Arnold,  9,  10 

hot  air,  7,  17 

steam,  ix 

STERNBERG,  G.  M.,  177 
STITT,  E.  R.,  301,  318 
STOCKING,  W.  A.,  132 
Stolon,  100 
Straw,  decomposition  of,  248 

microorganisms  on,  271 
Streptococci  in  milk,  275 
Streptococcus  erysipelatus,  347 

pyogenes,  47,  97,  318,  347 
Slreptothrix,  242 

actinomyces,  97 
Subcutaneous    inoculation,     297, 

309 

Subdural  inoculation,  299,  300 
Substrate,  nutrient,  101 
Sucrase,  180,  182 
Sugar,  caramelization  of,  26 

for     demonstrating     gas     pro- 
duction, 46 

milk,  184 

muscle,  359 
Sugars  as  food,  167,  170 

complex  as  food,  167 

fermentation  of,  171,  172,  182, 
184 

inversion  of,  23 

simple,  as  food,  167 
Sulfur,  lead-blackening,  174 

reduction  of,  181,  190,  191 


420 


INDEX 


Sulfur,  reductase,  181 

Sunlight,  effect  of,  176,  208,  209, 

243 

SURFACE,  F.  M.,  329 
Surfaces,     polished,     sterilization 

of,  6 
Surgical     dressings,     sterilization 

of,  10 

Suture,  299,  300 
Swab,  preparation  of,  150 

use  of,  316 

Symbiosis,  45,  214,  215,  256 
Synaptase,  180 

Synthetic  media,  171,  248,  350 
Syphilis,  hemolysin  in,  325 
Syringe,  295,  309 
Syringes,  sterilization  of,  10 


Tannase,  180 

Tannin,  375 
enzyme  of,  180 

TARROZZI,  155,  156 

Teeth,  microflora  of,  150 

Temperature,  16,  198-203 
body,  47 

cardinal  points  of,  198,  200 
constant,  ix,  47,  49,  52 
degrees,  conversion  of,  377 
effect    on    microorganism    and 

spores,  199-203 
effect    on    pigment    formation, 

175 

for  sterilization,  8-11 
influence  on  keeping  quality  of 

milk,  278-281 
low,  for  sterilization,  8 
maximum,  155,  198,  199 
minimum,  155,  198,  199 
optimum,  155,  198,  199 
pressure  table,  376 
relations,  155 

Tenacula,  299 


Test  tubes,  cleaning  of,  2 

plugging  of,  16 
Tests,  immunity,  13 

Tetanus,  antitoxin  preparation 
of,  309,  310 

toxin,  preparation  of,  308 
Thermal  deathpoint,  200,  201 
Thermogenic,  154 
Thermometer,  clinical,  313 
Thermophilic  bacteria,  155 
Thiobacteria,  348 
Thiocapsa,  348 
Thiocystis,  348 
Thiodictyon,  348 
Thiopedia,  348    \ 
Thiosarcina,  348 
Thiospirillum,  348 
Thiothece,  348 
Thiothrix,  348 
THRESH,  J.  C.,  177,  2271 
Throat,  microflora  of,  150 
Thrombase,  181 
Thrombin,  181 
Thymol,  15 

Time  for  sterilization,  8-11 
Tissue,     diseased,     isolation     of 
pathogens  from,  301,  302 

sterile  for  oxygen   absorption, 
156 

vegetable,  sterile,  156 
Tissues,  connective,  156 

staining  of,  96 

subcutaneous,  297,  299 
Titration,  20-22 

calculations,  21,  22 
Titre,  21,  25,  112 
Toluol,  15 
Tonneau,  380 
Torula  rosea,  83,  114,  120,  175, 

176,  205 
Torulce,  114 

Toxin,  tetanus,  preparation  of,  308 
Toxins,  308-310 


INDEX 


421 


TRALLES,  377 

Transferring  cultures,  51,  59-61 

Tray,  operating,  309 

Trephine,  295,  300 

Trjetrop's  anaerobic  apparatus,  159 

Trichobacterince,  348 

Trichophyton  tonsurans,  149 

Tricresol,  304 

Trocar,  299 

Trypsase,  180 

Trypsin,  24,  180 

Tryptase,  180 

Tube  length  of  microscope,  65,  66, 

71 
Tubercle     bacteria,     method     of 

staining,  92,  93 
Tubercles,  staining  of,  92,  93 
Tuberculin,  preparation  of,  303, 

306 
Tubes,  fermentation,  cleaning  of,  4 

plugging  of,  18 

vacuum,  158 
Tumblers,  use  of,  47 
Tumors,  crown-gall,  258 
Turbidity  in  water,  237 
Turro's  anaerobic  tube,  161 
TYNDALL,  J.,  8-10,  147 

method  for  sterilization,  8-10, 

193,  217,  356,  358 
Typhoid  group,  362,  363 
Tyrosin,  181 
Tyrosinase,  181 

U 

Udder,  bacteria  in,  268-270 

infection  of,  270 
Ultramicioscope,  33 
Urea,  181,  185,  352,  353 

decomposition,  media  for,  352, 

353 

Urease,  178,  181,  185 
Urine,     isolation     of     pathogens 
from,  302 


Uschinsky's  solution,  172,  350 
Utensils,  sterilization  of,  6-11 


Vaccine,  antirabic,  preparation  of, 
13 

black-leg,  preparation  of,  307 
Vaccines,    bacterial,    pieparatiort 
of,  316-318 

preservation  of,  15 
Vacuum,  for  anaerobic   cultures, 

156,  158-160 
VAILLARD,  309 
VAN  DER  HEIDE,  C.  C.,  35 
VAN  SLYKE,  L.  L.  and  PUBLOW, 

C.  K.,  287 
Vaselin,  76,  157,  296 
Vegetables,  as  a  medium,  19,  20 

diseases  of,  291,  294 
Vein,  femoral,  298 

jugular,  298 

VERNON,  H.  M.,  168, 186, 188, 192 
Vignal's  tube,  158 
Vinegar,  bacteria,  192 

circular  on,  192 

fermentation,  183 

legal  standard  of,  192 

oxidase,  181 

pure  cultures  for,  192 

titration  of,  22 
Violet,  crystal,  361 

gentian,  87,  373 
Virulence,  295 
Virus,  attenuated,  307 

dialyzation  of,  13 

filterable,  313-315 

hog  cholera,  313-315 
Vitality,  157 
Vosges-Proskauer's  reaction,  363 

W 

WARD,  A.  R.,  268,  270,  281,  284 
WASHBURN,  R.  M.,  291 


422 


INDEX 


Wastes,    laboratory,    destruction 

of,  6,  7 

Watch,  stop,  74 
Water,  as  an  end  product,  183 
bacteria  isolated  from,  223-227 
bacteriological  analysis  of,  223- 

235 

'  distilled,  20 
filtered,  238-239 
in  milk,  23 

of  condensation,  35,  38,  39,  52 
on  shipboard,  treatment  of,  237 
purification  of,.  236-239 
sterilization  of,  by  heat,  8-12 
Wax-like  substances  in  bacteria, 

92 
Weight    of   bacteria,    calculation 

of,  153,  244 
WELLS,  LEVI,  285 
Welsbach  burner,  67 
WESBROOK,   F.   F.,   WHITTAKER, 
H.  A.  and  MOHLER,  B.  M. 
237 

Whey,  sour,  169,  349 
WHITTAKER,  H.  A.,  238 
Widal  test,  361 
WILEY,  H.  W.,  291 
Wine-making,  yeasts  used,  114 
Wines,  19 
Winogradski's  medium  for  nitrate 

formation,  173,  350,  351 
medium  for  symbiotic  nitrogen- 
fixation,  173,  350,  351 
WINSLOW,  C.  E.  A.,  153,  222,  227 
WINSLOW,  C.  E.  A.  and  BROWN, 
W,  W.,  222 


Wood  ashes,  355 
Woodhead's  flask,  159 
Woodwork,  sterilizing  of,  14 
Wool,  effect  of  sterilization  on,  7 
Working  distance,  69,  77 
Wort,  beer,  20,  46,  78 

titration  of,  22 

Wounds,  sterilization  of,  6,  14,  48 
Wright's  stain,  319 
Wrzosek,  157 


Xylol,  effect  on  enzymes,  187,  188 
use  of,  65,  66,   187,   188,  265, 
266,  372 


Yeast,  63,  78-80,  84,  97 

cake,  flora  of,  115,  116 

Fleischmann's  Qompressed,  114, 

190 
Yeasts,  budding  of,  79 

cultivated,  114 

enzymes  of,  182,  190,  191 

fermenting  power  of,  83,  84 

pseudo,  114 

resistance  to  heat,  202,  203 

study  of,  114-123 

wild,  114 


Ziehl-Nielson's  carbol-fuchsin,  374 
Zone  development  of  colonies,  164 
Zymase,  180 

lactic  acid  bacteria,  180,  184 
Zymogenic,  153 


JBT?'-^ 


UNIVERSITY  OF  CALIFORNIA  LIBRARY 


